U.S. patent number 9,611,506 [Application Number 14/939,153] was granted by the patent office on 2017-04-04 for reaction mixtures for forming cdna from an rna template.
This patent grant is currently assigned to QIAGEN, GMBH. The grantee listed for this patent is QIAGEN GMBH. Invention is credited to Holger Engel, Christian Korfhage, Martin Kreutz, Dirk Loeffert, Subrahmanyam Yerramilli.
United States Patent |
9,611,506 |
Engel , et al. |
April 4, 2017 |
Reaction mixtures for forming cDNA from an RNA template
Abstract
This invention relates to a process for synthesis of a cDNA in a
sample, in an enzymatic reaction, whereby the process comprises the
steps: simultaneous preparation of a first enzyme with
polyadenylation activity, a second enzyme with reverse
transcriptase activity, a buffer, at least one ribonucleotide, at
least one deoxyribonucleotide, an anchor oligonucleotide; addition
of a sample that comprises a ribonucleic acid; and incubation of
the agents of the previous steps in one or more temperature steps,
which are selected such that the first enzyme and the second enzyme
show activity. The invention further relates to a reaction mixture
that comprises a first enzyme with polyadenylation activity, a
second enzyme with reverse transcriptase activity, optionally a
buffer, optionally at least one ribonucleotide, optionally at least
one deoxyribonucleotide, and optionally an anchor oligonucleotide.
Moreover, the invention relates to a kit that comprises a
corresponding reaction mixture.
Inventors: |
Engel; Holger (Hilden,
DE), Yerramilli; Subrahmanyam (Clarksville, MD),
Kreutz; Martin (Germantown, MD), Loeffert; Dirk
(Duesseldorf, DE), Korfhage; Christian (Hilden,
DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
QIAGEN GMBH |
Hilden |
N/A |
DE |
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Assignee: |
QIAGEN, GMBH (Hilden,
DE)
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Family
ID: |
38669769 |
Appl.
No.: |
14/939,153 |
Filed: |
November 12, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160130634 A1 |
May 12, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12377457 |
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9217173 |
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PCT/EP2007/058369 |
Aug 13, 2007 |
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Foreign Application Priority Data
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Aug 14, 2006 [DE] |
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10 2006 038 113 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6806 (20130101); C12Q 1/6848 (20130101); C12Q
1/6806 (20130101); C12Q 2527/101 (20130101); C12Q
2521/131 (20130101); C12Q 2521/107 (20130101) |
Current International
Class: |
C12P
19/34 (20060101); C12Q 1/68 (20060101) |
Field of
Search: |
;435/6.12,91.1,91.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1763223 |
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Apr 2006 |
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CN |
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2002-505087 |
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Feb 2002 |
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JP |
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2006-504440 |
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Feb 2006 |
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JP |
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WO 99/43650 |
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Sep 1999 |
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WO |
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WO 2004/044239 |
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May 2004 |
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WO |
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WO 2005/064019 |
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Jul 2005 |
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WO |
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Other References
Ambros, Victor, "The Functions of Animal MicroRNAs," Nature, vol.
431, pp. 350-355, Sep. 16, 2004. cited by applicant .
Ambros, Victor, "MicroRNAs: Tiny Regulators with Great Potential,"
Cell, vol. 107, pp. 823-826, Dec. 28, 2001. cited by applicant
.
Balakin, Andrey G., "The RNA World of the Nucleus: Two Major
Families of Small RNAs Defined by Different Box Elements with
Related Functions," Cell, vol. 86, pp. 823-834, Sep. 6, 1995. cited
by applicant .
Botero, Lina M., et al., "Poly(A) Polymerase Modification and
Reverse Transcriptase PCR Amplification of Environment RNA,"
Applied and Environmental Microbiology, vol. 7, No. 3, pp.
1267-1276, Mar. 2005. cited by applicant .
Chen, Chun-Long, et al., "The High Diversity of snoRNAs in Plants:
Identification and Comparative Study of 120 snoRNA Genes from Oryza
sativa," Nucleic Acids Research, vol. 31, No. 10, pp. 2601-2613,
2003. cited by applicant .
Fu, Hanjiang, et al., "Identification of Human Fetal Liver miRNAs
by a Novel Method," FEBS Letters, vol. 579, pp. 3849-3854, 2005.
cited by applicant .
Hell, A., et al.: "Synthesis of DNAs Complementary to Human
Ribosomal RNAs Polyadenylated in Vitro", Biochemical et Biophysica
Acta, vol. 442, pp. 37-49, 1976. cited by applicant .
Ko, J.H., et al.: "RNA-Conjugated Template Switching RT-PCR Method
for Generating an Escherichia coli cDNA Library for Small RNAs",
Journal of Microbiological Methods, vol. 64, pp. 297-304, 2006.
cited by applicant .
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Technique," Science, vol. 241, pp. 1077-1080, Aug. 26, 1988. cited
by applicant .
Lee, Yoontae, et al., "The Nuclear RNase III Drosha Initiates
MicroRNA Processing," Nature, vol. 425, pp. 415-419, Sep. 25, 2003.
cited by applicant .
Lee, Yoontae, et al., "MicroRNA Maturation: Stepwise Processing and
Subcellular Localization," The EMBO Journal, vol. 21, No. 17, pp.
4663-4670, 2002. cited by applicant .
Liu, Dongyu, et al., "Rolling Circle DNA Synthesis: Small Circular
Oligonucleotides as Efficient Templates for DNA Polymerases," J Am
Chem Soc, vol. 118, No. 7, pp. 1587-1594, 1996. cited by applicant
.
Lao, et al., "Multiplexing RT-PCR for the detection of multiple
miRNA Species in small samples" Biochemical and Biophysical
Research Communications, 343, (2006), 85-89. cited by applicant
.
Martin, G., and W. Keller, "Tailing and 3'-end Labelling of RNA
with Yeast Poly(A) Polymerase and Various Nucleotides," RNA, vol.
4, pp. 226-230, 1998. cited by applicant .
Maxwell, E. S., and M. J. Fournier, "The Small Nucleolar RNAs," Ann
Rev Biochem, vol. 35, pp. 897-934, 1995. cited by applicant .
Olsen, Gary J., et al., "Microbial Ecology and Evolution: A
Ribosomal RNA Approach," Ann Rev Microbiol, vol. 40, pp. 337-365,
1986. cited by applicant .
Sano, Hiroshi, and Gunter Feix, "Terminal Riboadenylate Transferase
from Escherichia coli," Eur J Biochem, vol. 71, pp. 577-583, 1976.
cited by applicant .
Shi, Rui, and Vincent L. Chiang, "Facile Means for Quantifying
MicroRNA Expression by Real-Time PCR," BioTechniques, vol. 39, No.
4, pp. 519-524, 2005. cited by applicant .
Walker, G. Terrance, et al., "Strand Displacement Amplification--an
Isothermal, in vitro DNA Amplification Technique," Nucleic Acids
Research, vol. 20, No. 7, pp. 1691-1696, 1992. cited by applicant
.
Wang, Jia-Fu, et al., "Identification of 20 MicroRNAs from Oryza
sativa," Nucleic Acids Research, vol. 32, No. 5, pp. 1688-1695,
2004. cited by applicant .
Wiedmann, M., et al., "Ligase Chain Reaction (LCR)--Overview and
Applications," Genome Research, vol. 3, pp. S51-S64, 1994. cited by
applicant .
International Search Report of PCT/EP2007/058369 (Dec. 12, 2007).
cited by applicant .
Japanese Office Action, Application No. 2009-524183, dated Nov. 20,
2012. cited by applicant .
Restriction Requirement issued in U.S. Appl. No. 12/377,457, mailed
Nov. 21, 2014. cited by applicant .
Non-Final Office Action issued in U.S. Appl. No. 12/377,457, mailed
Feb. 25, 2015. cited by applicant .
Final Office Action issued in U.S. Appl. No. 12/377,457, mailed on
Jul. 17, 2015. cited by applicant .
Notice of Allowance issued in U.S. Appl. No. 12/377,457. cited by
applicant.
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Primary Examiner: Horlick; Kenneth
Attorney, Agent or Firm: Seyfarth Shaw LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. application Ser.
No. 12/377,457, filed Feb., 13, 2009, which issued as U.S. Pat. No.
9,217,173 on Dec. 22, 2015, which is a U.S. National Phase entry of
PCT/EP2007/058369, filed Aug. 13, 2007, which claims priority to
German Patent Application No. 10 2006 038 113.0, filed Aug. 14,
2006, the disclosures of each of which are incorporated by
reference herein in their entireties.
Claims
What is claimed is:
1. A reaction mixture comprising: a. RNA; b. a first enzyme with
polyadenylation activity, c. a second enzyme with reverse
transcriptase activity; d. a buffer; e. at least one
ribonucleotide; f. at least one deoxyribonucleotide comprising: i.
a modification selected from the group consisting of a
biotinylation, a digoxigenin, and a hapten; or ii. a label selected
from the group consisting of a radioactive label and a fluorescent
label; and g. an anchor olignucleotide comprising a poly(T)
sequence capable of being extended to form cDNA using the RNA as a
template.
2. The reaction mixture of claim 1, wherein the RNA is selected
from the group consisting of prokaryotic RNA, eukaryotic RNA, viral
RNA, archae RNA, miRNA, snoRNA, mRNA, tRNA, non-polyadenylated RNA,
rRNA and mixtures thereof.
3. The reaction mixture of claim 1, wherein the anchor
oligonucleotide comprises a 5'-tail sequence.
4. The reaction mixture of claim 3, wherein the anchor
oligonucleotide has a length of between 6 and 150 nucleotides, and
optionally comprises an anchor sequence on the 3'-end.
5. The reaction mixture of claim 4, wherein the anchor
oligonucleotide is selected from the group consisting of a
deoxyribonucleic acid (DNA), a peptide-nucleic acid (PNA), a locked
nucleic acid (LNA), a phosphorus thioate-deoxyribonucleic acid, a
cyclohexene-nucleic acid (CeNA), an N3'-P5'-phosphorus amidate (NP)
and a tricyclo-deoxyribonucleic acid (tcDNA).
6. The reaction mixture of claim 1, wherein the at least one
ribonucleotide is selected from the group consisting of
adenosine-5'-triphosphate, and adenosine-5'-triphosphate with a
base analog, wherein the ribonucleotide is optionally modified or
labeled.
7. The reaction mixture of claim 1, wherein the deoxyribonucleotide
is selected from the group consisting of
deoxyadenosine-5'-triphosphate (dATP), deoxythymine-5'-triphosphate
(dTTP), deoxycytosine-5'-triphosphate (dCTP),
deoxyguamine-5'-triphosphate (dGTP), and
deoxyuracil-5'-triphosphate (dUTP).
8. The reaction mixture of claim 1, wherein the radioactive label
is selected from the group consisting of .sup.32P, .sup.33P,
.sup.35S, and .sup.3H.
9. The reaction mixture of claim 1, wherein the fluorescent label
is selected from the group consisting of fluorescein isothiocyanate
(FITC), 6-carboxyfluorescein (FAM), xanthene, rhodamine,
6-carboxy-2',4',7',4,7-hexachlorofluorescein,
6-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein,
N,N,N',N'-tetramethyl-6-carboxyrhodamine, 6-carboxy-X-rhodamine,
5-carboxyrhodamine-6G, 6-carboxyrhodamine-6G, rhodamine 110,
coumarins, umbelliferones, benzimides, phenanthridines, ethidium
bromides, acridine dyes, carbazole dyes, phenoxazine dyes,
porphyrine dyes, polymethine dyes; cyanine dyes, Cy3, Cy5, Cy7,
quinoline dyes and alexa dyes.
10. The reaction mixture of claim 1, wherein the concentration of
the deoxyribonucleotide is at least 0.01 mmol in the reaction
mixture and at most 10 mmol in the reaction mixture.
11. The reaction mixture of claim 10, wherein the concentration of
the deoxyribonucleotide is at least 0.2 mmol to at most 2 mmol.
12. The reaction mixture of claim 1, wherein the buffer has a pH of
6 to 10 and comprises Mg2+ ions.
13. The reaction mixture of claim 1, wherein the enzyme with
polyadenylation activity is selected from the group consisting of
an enzyme of prokaryotic origin, an enzyme of eukaryotic origin, an
enzyme of viral origin, an enzyme of archae origin and an enzyme of
plant origin.
14. The reaction mixture of claim 13, wherein the enzyme with
polyadenylation activity is selected from the group consisting of a
poly(A)-polymerase from Escherichia coli, a poly(A)-polymerase from
yeast, a poly(A)-polymerase from cattle, a poly(A)-polymerase from
frogs, and a human poly(A)-polymerase.
15. The reaction mixture of claim 1, wherein the enzyme with
reverse transcriptase activity is selected from the group
consisting of an enzyme from a virus, an enzyme from a bacteria, an
enzyme from an archae bacteria, an enzyme from a eukaryote and an
enzymes from a thermostable organism.
16. The reaction mixture of claim 15, wherein the enzyme with
reverse transcriptase activity is selected from the group
consisting of HIV Reverse Transcriptase, M-MLV Reverse
Transcriptase, EAIV Reverse Transcriptase, AMV Reverse
Transcriptase, Thermus thermophilus DNA polymerase 1, and M-MLV
RNAse H.
17. The reaction mixture of claim 1, further comprising a
thermostable enzyme with DNA-synthesis activity, and at least one
oligonucleotide for specific detection of cDNA.
Description
The instant application contains a Sequence Listing which has been
submitted via EFS-Web and is hereby incorporated by reference in
its entirety. Said ASCII copy, created on Mar. 30, 2012, is named
051612US1.txt and is 10,991 bytes in size.
BACKGROUND OF THE INVENTION
The invention relates to the field of molecular biology as well as
the research in this field but also the human as well as non-human
diagnosis.
The analysis of non-polyadenylated RNA molecules, such as, for
example, bacterial RNAs or small RNAs, such as the so-called
microRNAs (miRNAs), is made with difficulty and requires special
processes. A possible process was recently described in the
literature. This process comprises several enzymatic steps that are
connected in succession, i.e., first a "tailing" of the RNA with
poly-(A)-polymerase and a suitable substrate, typically ATP, is
performed. Then, the poly-(A)-reaction is stopped, and the reaction
product is purified. Then, the generated poly-(A)-RNA is added in a
reverse transcriptase reaction and is converted with suitable
primers into cDNA.
TECHNICAL FIELD
The performance of these two enzymatic reactions that are connected
in succession is expensive in the implementation and has a number
of error sources, for example input of nucleases, loss of material
or pipetting errors.
microRNAs (miRNAs) vary in size from about 20 to 25 nucleotides and
represent a new family of non-coding RNAs.
They are processed via a so-called "Hairpin Precursor" and can play
a role as negative regulators in the gene expression. They thus
adjust a number of genes downward (Ambros, V., 2001, MicroRNA's:
Tiny Regulators with Great Potential, Cell 107, 823-826). miRNAs
are first transcribed as long, "primary transcripts" (they are also
referred to as primary miRNAs) (Lee, Y., Jeon, K. et al., 2002,
MicroRNA Maturation: Stepwise Processing Subcellular Localisation,
Embo J. 21, 4663-4670). These "primary transcripts" are then
shortened, whereby the length resulting therefrom is in about 70
nucleotides. So-called "stem-loop structures" are produced; they
are also referred to as "pre-miRNAs." Pre-miRNAs are exported in
the cytoplasm. The exporting enzyme is named Exportin-5. They are
further processed here, and in this way, an approximately
22-nucleotide-long, mature miRNA molecule is produced (Lee, Y., et
al., 2003, The Nuclear RNA's III Drosha Initiates microRNA
Processing, Nature 425, 415-419). The most recent studies have
proposed that miRNAs play an important role in the development and
differentiation. In principle, microRNAs can have a regulating
action in two different ways. In plants, miRNAs complement with
their corresponding mRNAs by exact complementarity. This leads to a
destruction of the target-mRNA by a mechanism that comprises RNA
interference (RNAi). In animals, miRNAs prevent gene expression by
a mechanism, which comprises Lin-4 and Let-7. Here, the miRNAs are
not exactly complementary to their corresponding mRNAs, but they
prevent the synthesis and function of the proteins (Ambros, V.,
2004, The Functions of Animal microRNAs, Nature, 431, 350-355).
Because of the decisive role that the only recently discovered
miRNAs play, their detection or analysis is of decisive
importance.
In eukaryotes, the synthesis of the 18s, 5.8s and 25/28s rRNAs
comprises the processing in modifications of so-called
precursor-rRNAs (pre-rRNA) in the nucleolus. This complex course of
the rRNA biogenesis comprises many small so-called "small nucleolar
RNAs" (snoRNA), which accumulate in the nucleolus. They do this in
the form of so-called small nucleolar ribonucleo protein particles
(snoRNPs) (Maxwell, E. S. et al., 1995, The Small Nucleolar RNAs,
Annual Review Biochem, 35, 897-934).
All snoRNAs that are characterized to date, with the exception of
the RNase MRP, fall into two families. The latter are the box c/D
and box h/ACA slow RNAs, which can be distinguished by sequence
motifs common thereto (Ballakin, A. D. et al., 1996, The RNA World
of the Nucleolus: Two Major Families of Small Nucleolar RNAs
Defined by Different Box Elements with Related Functions, Cell, 86,
823-834). The genomic organization of the snoRNA genes has a large
diversity in various eukaryotes.
In vertebrates, most snoRNAs are introduced within Intrans via
"host genes." Exceptions such as U3 are independently transcribed.
In yeast, there are snoRNAs that are introduced into Intrans, but
the majority of the snoRNAs are transcribed as single genes with a
separate promoter. Clustered snoRNA genes are transcribed upstream
by common promoters. Based on the small sizes and the deficient
polyadenylation, the detection or the analysis of snoRNAs is a
molecular-biological challenge.
The PCR is a frequently-used instrument for the study of microbial
organisms and is also used, i.a., to analyze 16S rRNA genes.
However, the discovery of new genes in microbial samples is limited
by the only conditionally possible synthesis of primers.
Thus, primers are derived for 16S RNA genes from those sequences
that are already known from cultivated microbes (Olson, D. J.,
1986, Microbial Ecology and Evolution:
A Ribosomal RNA Approach, Annu. Rev. Microbial. 40: 337-365). Based
on the systematics that there is recourse to sequences that are
already known namely for the extraction of 16S rRNA genes from
organisms that are unknown to date, it is probable that the
microbial diversity is greatly underestimated and also not
isolated.
Just as the 16S rRNA molecules can only be isolated with
difficulty, prokaryotic mRNA molecules can be isolated with
difficulty owing to a lack of knowledge of the sequence and in
particular owing to a lack of poly-A-tail.
The prior art knows a 2-stage process. In this process, an RNA
molecule is reacted with the aid of the enzyme poly-A-polymerase
and the substrate adenosine triphosphate, such that a
polyadenylated ribonucleic acid molecule is produced. This thus
polyadenylated ribonucleic acid molecule is purified in an
additional step before a reverse transcription takes place in a
third step. Reverse transcription is appropriated in the
polyadenylated tail, whereby a homopolymer oligonucleotide in
general attaches a poly-oligonucleotide to the polyadenylated RNA
tail in a complementary manner. The 3'-end of the
poly-T-oligonucleotide is now used by the polymerase to produce a
deoxyribonucleic acid strand, which is complementary to the
existing ribonucleic acid strand. The thus produced strand is named
"first strand of cDNA." This cDNA can be used in a PCR reaction,
whereby it results in the use of either random primers or else
specific primers to generate an amplificat. Shi et al. teaches
especially the miRNA detection via an oligo-dT adapter-primer,
whereby an adapter of the specific primer is used in the PCR (Shi,
R., and Chiang, V. L. (Shi, R. et al., Facile Means for Quantifying
microRNA Expression by Real-Time PCR, Biotechniques, 2005, 39,
519-25).
This only recently published process has decisive drawbacks
relative to the above-mentioned special ribonucleic acid
molecules.
Thus, the two-stage process in general may involve an introduction
of contaminants. The purification step leads to losses of rare
RNAs. The two-stage process requires an inactivation of the first
enzyme as well as an incubation time for the first enzyme and the
second enzyme, which together results in a very great time
expenditure. In addition, the two-stage process has the drawback
that a danger of confusion of samples can then occur when two or
more samples are treated at the same time. As is known from the
prior art, ribonucleic acids are relatively sensitive as far as the
attack of nucleases is concerned. The two-stage process, in
particular the step of purification after the first process,
involves the danger that nucleases are introduced. Ultimately, two
or more steps always lead to the fact that the danger of pipetting
errors increases.
SUBJECT OF THE INVENTION
It is thus the object of this invention to prepare a process that
makes possible the cDNA synthesis, prevents contaminants as much as
possible, is less time-consuming, minimizes the danger of confusion
of the samples, minimizes the danger of the introduction of
nucleases, and finally excludes the danger of pipetting errors as
much as possible.
This object is achieved by a process for synthesis of a cDNA in a
sample, in an enzymatic reaction, whereby the process comprises the
following steps:
(a) Simultaneous preparation of a first enzyme with polyadenylation
activity, a second enzyme with reverse transcriptase activity, a
buffer, at least one ribonucleotide, at least one
deoxyribonucleotide, an anchor oligonucleotide, (b) addition of a
sample that comprises a ribonucleic acid, and (c) incubation of the
agents of steps (a) and (b) in one or more temperature steps, which
are selected such that the first enzyme and the second enzyme show
activity.
Up until today, there have been concerns about a combination of the
enzymatic polyadenylation and the reverse transcriptase being
technically possible. This is shown in that even more recently,
i.e., after discovery of microRNAs and snoRNAs, which represent a
special molecular-biological challenge as regards analysis and
isolation, the enzymatic reactions were always performed in
succession (Want, J. F., et al., Identification of 20 microRNAs
from Oryza sativa, Nucleic Acid Res., 2004, 32, 1688-95; Shi, R.
and Chiang, V. L., Facile Means for Quantifying microRNA Expression
by Real-Time PCR, Biotechniques, 2005, 39, 519-25; Fu, H., et al.,
Identification of Human Fetal Liver miRNAs by a Novel Method; FEBS
Lett, 2005, 579, 3849-54; Chen, C. L. et al., The High Diversity of
snoRNAs in Plants: Identification and Comparative Study of 120
snoRNA Genes from Oryza Sativa, Nucleic Acids Res, 2003, 31,
2601-13; Botero, L. M. et al., Poly(A) Polymerase Modification and
Reverse Transcriptase PCR Amplification of Environmental RNA, Appl.
Environ Microbial, 2005, 71, 1267-75). Surprisingly enough, both
processes, i.e., the polyadenylation and reverse transcription,
have already been known to one skilled in the art for a long time
(Sano, H., and Feix, G., Terminal Riboadenylate Transferase from
Escherichia coli. Characterization and Application, Eur. J.
Biochem., 1976, 71, 577-83). In general, according to the
poly-A-tailing step, one skilled in the art has purified the
reaction product (Shi, R., et al., Facile Means for Quantifying
microRNA Expression by Real-Time PCR, Biotechniques, 2005, 39,
519-25). The reason for this lies both in the clearly different
compositions of the reaction buffers and the substrates that are
required for the reaction.
Another subject of this invention is to prepare a simple process
that makes the cDNA synthesis possible and couples this reaction
optionally with a third enzymatic reaction, which allows the
specific detection of the generated cDNA in the same reaction
vessel. By a very simple handling, this "3-in-1" process shows
special advantages when a large number of samples are to be
analyzed in one or a few analytes. The reason is that, e.g.,
coupled to a real-time PCR, a very quick and simple process shows a
large number of samples to be analyzed. Additional handling steps
and contaminations are to be prevented as much as possible, by
which it is less time-consuming, the danger of confusion of the
samples is minimized, the danger of the introduction of nucleases
is minimized, and ultimately, the danger of pipetting errors is
excluded as much as possible.
The object of the "3-in-1" reaction is achieved by a process for
the synthesis of a cDNA in a sample, in an enzymatic reaction,
followed by another enzymatic reaction, optionally an
amplification, optionally coupled to the detection, either in
real-time during the amplification or downstream, whereby the
process comprises the following steps: (a) simultaneous preparation
of a first enzyme with polyadenylation activity, a second enzyme
with reverse transcriptase activity, a buffer, at least one
ribonucleotide, at least one deoxyribonucleotide, an anchor
oligonucleotide, at least a third enzyme with nucleic
acid-synthesis activity, at least one primer, and optionally a
probe, (b) addition of a sample that comprises a ribonucleic acid,
and (c) incubation of the agents of steps (a) and (b) in one or
more temperature steps, which are selected such that the first and
second enzyme show activity, and optionally the third enzyme is
active or inactive. Optionally one or more temperature steps
follow, in which the first and second enzymes are less active or
inactive, and the third enzyme is active.
The substrate of the poly-(A)-polymerase that is used in vivo is
adenosine triphosphate (ATP). For some poly-(A)-polymerases, it was
shown that even attaching a short tail to other NTPs as a substrate
can be possible (Martin, G., and Keller, W., Tailing and 3'-End
Labeling of RNA with Yeast Poly(A) Polymerase and Various
Nucleotides, RNA, 1998, 4, 226-30).
Surprisingly enough, the inventors of this invention had discovered
that it is possible, under certain requirements, to be able to
execute the two still very different enzymatic reactions
simultaneously in one reaction vessel. In a preferred embodiment of
the invention, the sample is a ribonucleic acid, which is selected
from the group that comprises prokaryotic ribonucleic acids,
eukaryotic ribonucleic acids, viral ribonucleic acids, ribonucleic
acids whose origin is an archae-organism, microribonucleic acids
(miRNA), small nucleolar ribonucleic acids (snoRNA), messenger
ribonucleic acid (mRNA), transfer-ribonucleic acids (tRNA),
non-polyadenylated ribonucleic acids in general, as well as
ribosomal ribonucleic acids (rRNA). Moreover, a mixture of two or
more of the above-mentioned ribonucleic acids. In the sample, of
course, poly-A RNA can also already be contained.
In an especially preferred embodiment of this invention, the sample
is a ribonucleic acid, which is selected from the group that
comprises prokaryotic ribonucleic acids, miRNA, snoRNA and rRNA. In
the most preferred embodiment of this invention, the sample
comprises a ribonucleic acid, which is selected from the group that
comprises miRNA and snoRNA. Other mixed samples that consist of
different amounts of ribonucleic acids of different types
associated with other substances are preferred.
In addition, the inventors of this invention have discovered that
it is possible, under certain conditions, to be able to execute the
two still very different enzymatic reactions simultaneously in one
reaction vessel as well as to couple the latter in addition to a
third enzymatic reaction for specific detection of the generated
cDNA, which is preferably a nucleic acid-synthesis activity. In a
preferred embodiment of the invention, the sample is a ribonucleic
acid, which is selected from the group that comprises prokaryotic
ribonucleic acids, eukaryotic ribonucleic acids, viral ribonucleic
acids, ribonucleic acids whose origin is an archae-organism,
microribonucleic acids (miRNA), small nucleolar ribonucleic acids
(snoRNA), messenger ribonucleic acid (mRNA), transfer-ribonucleic
acids (tRNA), and non-polyadenylated ribonucleic acids in general,
as well as ribosomal ribonucleic acids (rRNA). Moreover, a mixture
of two or more of the above-mentioned ribonucleic acids. In the
sample, of course, poly-A RNA can also already be contained.
In an especially preferred embodiment of this invention, the sample
is a ribonucleic acid that is selected from the group that
comprises prokaryotic ribonucleic acids, miRNA, snoRNA and rRNA. In
the most preferred embodiment of this invention, the sample
comprises a ribonucleic acid, which is selected from the group that
comprises miRNA and snoRNA. Other mixed samples that consist of
different amounts of ribonucleic acids of different types
associated with other substances are preferred. Based on these
advantages of the process according to the invention, the inventors
could show that it is possible to prepare and to characterize
miRNAs efficiently and without contamination.
In one embodiment of the invention, the anchor oligonucleotide is a
homopolymer oligonucleotide, which is selected from the group that
comprises a poly-(A)-oligonucleotide, poly-(C)-oligonucleotide,
poly-(T)-oligonucleotide, poly-(G)-oligonucleotide,
poly-(U)-oligonucleotide, poly-(A)-oligonucleotide additionally
comprising a 5'-tail, poly-(C)-oligonucleotide additionally
comprising a 5'-tail, poly-(T)-oligonucleotide additionally
comprising a 5'-tail, poly-(G)-oligonucleotide additionally
comprising a 5'-tail and poly-(U)-oligonucleotide additionally
comprising a 5'-tail. A poly-(T)-oligonucleotide, which optionally,
as already explained above, additionally can have a 5'-tail, is
preferred.
The anchor oligonucleotide according to the invention is generally
between 6 and 75 nucleotides long. However, it can be up to about
150 nucleotides long. If the anchor oligonucleotide is synthetic,
the maximum length follows from the technical limitations of the
DNA synthesis. The anchor oligonucleotide optionally comprises a
5'-tail and/or an anchor sequence. A 5'-tail is an additional
nucleotide sequence on the 5'-end of the oligonucleotide, which is
used, for example, to introduce a cloning sequence, primer and/or
probe-binding sites or any other sequence. The identification of
suitable sequences for the 5'-tail is possible for one skilled in
the art based on the requirements of the respective
application.
On the 3'-end of the anchor oligonucleotide, an additional anchor
sequence, typically with a length of one to five additional
nucleotides, can be contained. The anchor sequence can have a
length of at least one base, whereby the first position in a
preferred embodiment is a degenerated base, which contains all
bases except for the base that is used in the homopolymer portion
of the anchor oligonucleotide. After that, other bases can follow.
The latter can also be degenerated. In one preferred embodiment
here, the use of N wobbles is useful, whereby N=A, C, G, T or
corresponding analogs.
Normally, the anchor oligonucleotide is a deoxyribonucleic acid
(DNA). The anchor oligonucleotide, however, can also be a peptide
nucleic acid (PNA). Locked nucleic acids (LNA), phosphorus
thioate-deoxyribonucleic acids, cyclohexene-nucleic acids (CeNA),
N3'-P5'-phosphoramedites (NP), and tricyclo-deoxyribonucleic acids
(tcDNA) are also possible. An anchor oligonucleotide, which is a
deoxyribonucleic acid (DNA), is preferred, however. Mixtures of RNA
and DNA or one or more of the modified nucleic acids or analogs, as
well as other modifications, such as corresponding base analogs,
which are able to hybridize with RNA or DNA under the selected
conditions, are possible. In one especially preferred embodiment,
the anchor oligonucleotide is a poly-(T)-oligonucleotide, which
additionally comprises a 5'-tail, is a deoxyribonucleic acid, is
15-150 nucleotides long, and is present as a mixture. On the 3'-end
of the anchor oligonucleotide, an additional anchor sequence
typically with a length of one to five additional nucleotides can
be contained. The anchor sequence can have a length of at least one
base, whereby the first position in a preferred embodiment is a
degenerated base, which contains all bases except for the base that
is used in the homopolymer portion of the anchor oligonucleotide.
After that, other bases can follow. The latter can also be
degenerated. Here, in a preferred embodiment, the use of N wobbles
is useful, whereby N=A, C, G, T or corresponding analogs.
By way of example, the following anchor oligonucleotides according
to the invention can be mentioned:
TABLE-US-00001 Example 1 (SEQ ID NO: 10): 5' TGG AAC GAG ACG ACG
ACA GAC CAA GCT TCC CGT TCT CAG CC (T)xVVN 3' Example 2 (SEQ ID NO:
11): 5' AACGAGACGACGACAGAC(T)x VN 3' Example 3 (SEQ ID NO: 12): 5'
AACGAGACGACGACAGAC(T)x V 3' Example 4 (SEQ ID NO: 13): 5'
AACGAGACGACGACAGAC(T)x N 3' Example 5 (SEQ ID NO: 14): 5'
AACGAGACGACGACAGAC(T)x NN 3' Example 6 (SEQ ID NO: 15): 5'
AACGAGACGACGACAGAC(T)x VNN 3' Example 7 (SEQ ID NO: 16): 5'
AACGAGACGACGACAGAC(T)x VNNN 3' Example 8 (SEQ ID NO: 17): 5'
AACGAGACGACGACAGAC(T)x NNN 3' Example 9 (SEQ ID NO: 18): 5' TGG AAC
GAG ACG ACG ACA GAC CAA GCT TCC CGT TCT CAG CC(T)x VN 3'' Example
10 (SEQ ID NO: 19): 5' TGG AAC GAG ACG ACG ACA GAC CAA GCT TCC CGT
TCT CAG CC(T)x VNN 3'
X is preferably 10 to 30 bases. V and N are from the single letter
code for degenerated bases, V=A, C, G; N=A, C, G, T.
The identification of other suitable 5'-tail sequences is possible
to one skilled in the art.
The optional 5'-tail comprises additional 1-100 nucleotides, which
can be used for the following analyses. Thus, in a preferred
embodiment, the 5'-tail can contain the binding sequence for an
oligonucleotide, such as, e.g., one or more DNA probes and/or one
or more PCR primers. The sequences that are used for the 5'-tail
are preferably selected such that the latter are compatible with
the process according to the invention. This comprises, e.g., the
selection of those sequences that do not cause any undesirable
secondary reactions, both in the process according to the invention
and in the subsequent analysis process.
According to the invention, anchor oligonucleotides are shown in
FIG. 12.
In principle, the enzymatic reaction according to the invention can
take place on a vehicle or in a container, i.e., the reaction can
take place in a reaction vessel. Such a reaction vessel can be a
reaction tube or, for example, a microtiter plate. The reaction can
take place on a chip. If it takes place on a chip, one or more
components can be immobilized. The reaction can take place on a
test strip or in a microfluidic system. The most varied embodiments
relative to the vehicle or container are known to one skilled in
the art.
In a preferred embodiment, the ribonucleotide is an
adenosine-5'-triphosphate, a thymidine-5'-triphosphate, a
cytosine-5'-triphosphate, a guanine-5'-triphosphate and/or a
uracil-5'-triphosphate. The ribonucleotide can also be a base
analog. The ribonucleotide can be modified or labeled. In
principle, it is essential that the ribonucleotide can be reacted
by the enzyme in the polyadenylation activity as substrate.
The deoxyribonucleotide according to the invention can be selected
from the group that comprises a deoxyadenosine-5'-triphosphate
(dATP), a deoxythymine-5'-triphosphate (dTTP), a
deoxycytosine-5'-triphosphate (dCTP), a
deoxyguanosine-5'-triphosphate (dGTP), a
deoxayuracil-5'-triphosphate (dUTP) as well as modified
deoxyribonucleotides and labeled deoxyribonucleotides. Applications
are also conceivable in which in addition to or in exchange, one or
more deoxyribonucleotides, which contain a universal base or a base
analog, are used. It is essential for the implementation of the
invention that the deoxyribonucleotides that are used allow a cDNA
synthesis.
According to the invention, it is preferred if dATP, dCTP, dTTP,
and dGTP are present together as a mixture.
According to the invention, deoxyuracil-5'-triphosphate can also be
used in the mixture. This can be combined with an enzymatic
reaction that takes place after the actual reaction and that uses
the uracil-DNA-glycosilase and cannot degrade further used
enzymatically produced reaction product.
If a deoxyribonucleotide is labeled, the labeling can be selected
from the group that comprises .sup.32P, .sup.33P, .sup.35S,
.sup.3H; a fluorescent dye, such as, for example, fluorescein
isothiocyanate (FITC), 6-carboxyfluorescein (FAM), xanthene,
rhodamine, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX),
6-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein (JOE),
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA),
6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5),
6-carboxyrhodamine-6G (RG6), rhodamine 110; coumarins, such as
umbelliferones; benzimides, such as Hoechst 33258; phenanthridines,
such as Texas Red, ethidium bromides, acridine dyes, carbazole
dyes, phenoxazine dyes, porphyrine dyes, polymethine dyes; cyanine
dyes, such as Cy3, Cy5, Cy7, BODIPY dyes, quinoline dyes and alexa
dyes. Other labels such as the inclusion of biotin or one or more
haptens, such as, e.g., digoxigenin, which allow a direct or
indirect detection of the nucleic acid. Indirect detection, such
as, e.g., via antibodies, which in turn, e.g., enzymatic detection
via an enzyme coupled to an antibody. Also, via the introduction of
nanoparticles, which are coupled to, e.g., antibodies or an
affinity ligand, an indirect detection is possible.
A modification of the deoxyribonucleotide can also be carried out
via the 5'-phosphate, which allows a simpler cloning. By including
reactive groups, such as, e.g., an amino linker (also biotin), the
deoxyribonucleotide can be, e.g., immobilized, or a direct or
indirect detection can be made available.
Especially preferred modifications are selected from the group that
comprises fluorescence dyes, haptens, 5'-phosphate, 5'-biotin, and
5'-amino linkers.
According to the invention, the concentration of a
deoxyribonucleotide in the reaction is at least 0.01 mmol and at
most 10 mmol. This concentration information is the concentration
of the individual deoxyribonucleotide. In one of the preferred
embodiments, the deoxyribonucleotides, in each case dATP, dCTP,
dGTP and dTTP, are present at a concentration of 0.2 mmol to 2
mmol. This concentration information is the concentration of the
individual deoxyribonucleotide in the mixture. In an especially
preferred embodiment of the invention, the individual
deoxyribonucleotide, dATP, dCTP, dGTP and dTTP, is present at a
concentration of 0.5 mmol in each case.
Surprisingly enough, the inventors have determined that the
one-step enzyme reaction, as it is the subject of this invention,
can take place in a narrow buffer-pH range of 6 to 10 with the
presence of magnesium ions (Mg.sup.2+). Thus, a pH of 6 to 10 is
present in a preferred embodiment.
In an especially preferred embodiment, the buffer according to the
invention has a pH of 6.8 to 9.
In another embodiment of the invention according to the invention,
the buffer according to the invention comprises additional ions,
which can be selected from the group that comprises Mn.sup.2+, K+,
NH.sup.4+, and Na+.
Buffers according to the invention contain, for example,
MgCl.sub.2, MgSO.sub.4, magnesium acetate, MnCl.sub.2, KCl,
(NH.sub.4)zS0.sub.4, NH.sub.4Cl, and NaCl. As buffer substances,
tris, tricine, bicine, HEPES, as well as other buffer substances
that are in the pH range according to the invention or mixtures of
two or more appropriate buffer substances are suitable.
A number of enzymes with polyadenylation activity are known to one
skilled in the art. According to the invention, the latter are
selected from the group that comprises enzymes of prokaryotic
origin, eukaryotic origin, viral origin and archae origin as well
as also enzymes of plant origin.
A polyadenylation activity in terms of this invention is an
enzymatic activity that uses the 3'-end of a ribonucleic acid as a
substrate and is able to add enzymatic ribonucleotides,
specifically preferably at least 10 to 20 ribonucleotides, to this
3'-end in a suitable buffer. In a preferred embodiment, the enzyme
is an enzyme that is able to use adenosine-5'-triphosphate as a
substrate. According to the invention, the latter comprises enzymes
and reaction conditions that have a polyadenylation activity in
terms of the invention when using single-strand and double-strand
RNA, e.g., hairpin RNA, such as, e.g., pre-miRNA. Based on the RNA
to be analyzed, one skilled in the art can select enzymes and
reaction conditions such that either single-strand RNAs (e.g.,
mature miRNAs), or double-strand RNAs (e.g., pre-miRNAs) or both
are made available for analysis.
In general, a polyadenylation activity in terms of the invention is
a transcriptase activity.
In a preferred embodiment, the enzyme with polyadenylation activity
is an enzyme that is selected from the group that comprises
poly-(A)-polymerase from Escherichia coli, poly-(A)-polymerase from
yeast, poly-(A)-polymerase from cattle, poly-(A)-polymerase from
frogs, human poly-(A)-polymerase, and plant poly-(A)-polymerase.
Others are known to one skilled in the art or can be newly
identified by the analysis of homology in known
poly-(A)-polymerases. In an especially preferred embodiment, the
enzyme with polyadenylation activity is a poly-(A)-polymerase from
Escherichia coli.
The enzyme with reverse transcriptase activity according to the
invention is selected according to the invention from the group
that comprises enzymes from viruses, bacteria, archae-bacteria and
eukaryotes, in particular from thermostable organisms. These also
include, e.g., enzymes from Intrans, retrotransposons or
retroviruses. An enzyme with reverse transcriptase activity is an
enzyme, according to the invention, which is able to incorporate
deoxyribonucleotides in a complementary way in a ribonucleic acid
on the 3'-end of a deoxyoligonucleotide or ribooligonucleotide that
is hybridized on the ribonucleic acid under suitable buffer
conditions. This comprises, on the one hand, enzymes that of course
have this function but also enzymes that obtain such a function
only by changing their gene sequence, such as, e.g., mutagenesis,
or by corresponding buffer conditions.
Preferred is the enzyme with reverse transcriptase activity, an
enzyme that is selected from the group that comprises HIV Reverse
Transcriptase, M-MLV Reverse Transcriptase, EAIV Reverse
Transcriptase, AMY Reverse Transcriptase, Thermus thermophilus DNA
polymerase I, M-MLV RNAse H, Superscript, Superscript II,
Superscript Ill, MonsterScript (Epicenter), Omniscript, Sensiscript
Reverse Transcriptase (Qiagen), ThermoScript and Thermo-X (both
Invitrogen). According to the invention, enzymes can also be used
that as enzymes have reverse transcriptase only after a
modification of the gene sequence. A reverse transcriptase activity
that has elevated accuracy can also be used. By way of example,
e.g., AccuScript Reverse Transcriptase (Stratagene) can be
mentioned here. It is evident to one skilled in the art that even
the use of mixtures of two or more enzymes with reverse
transcriptase activity is possible.
It is known to one skilled in the art that most enzymes with
reverse transcriptase activity require a divalent ion. Thus, in a
preferred embodiment as already described above, a divalent ion is
present in those enzymes that require a divalent ion. Mg.sup.2+ and
Mn.sup.2 are preferred.
Preferred combinations of enzymes are HIV Reverse Transcriptase or
M-MLV Reverse Transcriptase or EAIV Reverse Transcriptase or AMY
Reverse Transcriptase or Thermus thermophilus DNA polymerase I or
M-MLV RNAse H, Superscript, Superscript II, Superscript III or
MonsterScript (Epicenter) or Omniscript Reverse Transcriptase
(Qiagen) or Sensiscript Reverse Transcriptase (Qiagen),
ThermoScript, Thermo-X (both Invitrogen) or a mixture of two or
more enzymes with reverse transcriptase activity and
poly-(A)-polymerase from Escherichia coli. In addition, HIV Reverse
Transcriptase or M-MLV Reverse Transcriptase or EAIV Reverse
Transcriptase or AMY Reverse Transcriptase or Thermus thermophilus
DNA Polymerase I or M-MLV RNAse H, Superscript, Superscript II,
Superscript III or MonsterScript (Epicenter) or Omniscript Reverse
Transcriptase (Qiagen) or Sensiscript Reverse Transcriptase
(Qiagen), ThermoScript, Thermo-X (both Invitrogen) or a mixture of
two or more enzymes with reverse transcriptase activity and
poly-(A)-polymerase from yeast.
It is known to one skilled in the art that high temperatures in
reverse transcription have the effect that problems with secondary
structures do not play a decisive role. Moreover, high temperatures
in certain enzymes have the effect that the specificity of reverse
transcription increases such that false pairs and false priming are
suppressed. Thus, a reverse transcriptase, which is thermophilic,
is used in an embodiment of this invention. An enzyme that has an
optimum nucleic acid synthesis activity at between 45.degree. C.
and 85.degree. C. is preferred, more preferred between 55.degree.
C. and 80.degree. C., and most preferred between 60.degree. C. and
75.degree. C. Thermus thermophilus (Tth) DNA polymerase I is
preferred.
If the enzyme with polyadenylation activity is a non-thermophilic
enzyme and the enzyme with reverse transcriptase activity is a
thermophilic enzyme, the process can be carried out in several
temperature steps according to the invention, whereby the first
temperature step allows a temperature to be used that is the
optimum temperature for the enzyme with polyadenylation activity,
and the second temperature step allows a temperature to be used
that is the optimum temperature for the enzyme with reverse
transcriptase activity.
If, for example, the AMY reverse transcriptase is used, the second
temperature step takes place at 42.degree. C., while the first
temperature step, which has primarily the activity of the enzyme
with polyadenylation activity, takes place at a temperature of
37.degree. C. Implementation at a constant temperature is also
possible, however.
One skilled in the art is able to select the temperatures so that
the respective enzyme activities have an impact. If, for example, a
combination of poly-(A)-polymerase from Escherichia coli
accompanied by DNA polymerase from Thermus thermophilus is used,
the course of the temperatures appears as follows: first, it is
incubated at 37.degree. C. and then at 55 to 70.degree. C.
According to the invention, a non-thermostable enzyme can thus be
combined with a thermostable enzyme. In this case, the temperature
steps then depend on which of the two enzymes has polyadenylation
activity. According to the invention, it is preferred that the
enzyme with reverse transcriptase reactivity be thermostable. In
the opposite case, and this is plausible to one skilled in the art,
it may be that by the incubation at a higher temperature in the
polyadenylation step, the enzyme with reverse transcriptase
activity is partially or completely inactivated. Thus, it is also
preferred if the two enzymes are thermostable.
In addition, it is known to one skilled in the art that the enzymes
are processive to very different degrees, so that one skilled in
the art can combine enzymes with different processivity in such a
way that templates of varying lengths are readily converted into
cDNA in different ways. By using corresponding amounts of the
respective enzymes, of one or more suitable incubation temperatures
and incubation times, it is possible for one skilled in the art to
achieve satisfactory results.
The process according to the invention preferably comprises
additional poly-(C)-polynucleotides. The process according to the
invention especially preferably comprises additional
poly-(C)-polyribonucleotides. Preferably, 1 ng to 300 ng of
poly-(C)-polyribonucleotides for each 20 .mu.l is incorporated,
preferably 10 ng to 150 ng of poly-(C)-polyribonucleotides for each
20 .mu.l of reaction is incorporated, especially preferably 25 ng
to 100 ng of poly-(C)-polyribonucleotides for each reaction is
incorporated, and most preferably 50 ng to 75 ng of
poly-(C)-polyribonucleotides for each 20 .mu.l of reaction is
incorporated.
The reaction according to the invention can comprise additional
reagents, such as, for example, volume excluder, a single-strand
binding protein, DTT and/or competitor nucleic acids.
If a volume excluder is used, the latter is selected from the group
that comprises dextran, and polyethylene glycol, and in EP
1411133A1, volume excluders according to the invention are
mentioned.
In a preferred embodiment, the competitor-nucleic acid is a
homopolymer ribonucleic acid, most preferably polyadenoribonucleic
acid. Examples are disclosed in U.S. Pat. No. 6,300,069.
The process according to the invention preferably comprises
additional poly-(C)-polynucleotides. The process according to the
invention especially preferably comprises additional
poly-(C)-polyribonucleotides. Preferably, 1 ng to 300 ng of
poly-(C)-polyribonucleotides is incorporated for each 20 .mu.l;
preferably 10 ng to 150 ng of poly-(C)-polyribonucleotides is
incorporated for each 20 .mu.l of reaction; especially preferably,
25 ng to 100 ng of poly-(C)-polyribonucleotides is incorporated for
each reaction; and most preferably 50 ng to 75 ng of
poly-(C)-polyribonucleotides is incorporated for each 20 .mu.l of
reaction.
It is obvious to one skilled in the art that it may be advantageous
to prevent the competitor-nucleic acid itself from being used as a
substrate for the poly-(A)-polymerase activity. A possible solution
is the blocking of the 3' OH group of the competitor-nucleic acid.
Corresponding solutions, such as, e.g., use of a 3' phosphate,
incorporation of a dideoxynucleotide or reverse bases, are known to
one skilled in the art.
It is also obvious to one skilled in the art that it is
advantageous to be able to prevent the competitor-nucleic acid
itself from being used as a substrate for the reverse transcriptase
activity. This can be ensured by selecting a competitor-nucleic
acid that cannot be converted into cDNA under the given reaction
conditions, e.g., since the primers that are used cannot hybridize
onto the latter. Another possible solution is the blocking of the
3' OH group of the competitor-nucleic acid. Corresponding
solutions, such as, e.g., use of a 3' phosphate, incorporation of a
dideoxynucleotide or reverse bases, are known to one skilled in the
art.
In addition, the invention relates to a reaction mixture that
comprises a first enzyme with polyadenylation activity, a second
enzyme with reverse transcriptase activity, optionally a buffer,
optionally at least one ribonucleotide, optionally at least one
deoxyribonucleotide and optionally one anchor oligonucleotide. The
anchor oligonucleotide preferably comprises a homopolymer portion,
an anchor sequence and/or a tail. The reaction mixture additionally
preferably comprises random primers. The additional use of random
primers has the advantage that even 5'-ends of long transcripts are
efficiently converted, which is important in quantitative analyses.
The reaction mixture can contain the same agents as are used for
the process according to the invention.
In an embodiment of the invention, the anchor oligonucleotide is a
homopolymer oligonucleotide, which is selected from the group that
comprises a poly-(A)-oligonucleotide, poly-(C)-oligonucleotide,
poly-(T)-oligonucleotide, poly-(G)-oligonucleotide,
poly-(U)-oligonucleotide, poly-(A)-oligonucleotide additionally
comprising a 5'-tail, poly-(C)-oligonucleotide additionally
comprising a 5'-tail, poly-(T)-oligonucleotide additionally
comprising a 5'-tail, poly-(G)-oligonucleotide additionally
comprising a 5'-tail and poly-(U)-oligonucleotide additionally
comprising a 5'-tail. Preferred is a poly-(T)-oligonucleotide,
which optionally in addition can have a 5'-tail as already
explained above.
The anchor oligonucleotide according to the invention is generally
between 6 and 75 nucleotides long. It can be up to about 150
nucleotides long, however. If the anchor oligonucleotide is
synthetic, the maximum length is produced from the technical
limitations of the DNA synthesis. The anchor oligonucleotide
optionally comprises a 5'-tail and/or an anchor sequence. A 5'-tail
is an additional nucleotide sequence on the 5'-end of the
oligonucleotide, which, for example, in this case is used to insert
a cloning sequence, primer and/or probe-binding sites, or any other
sequence. The identification of suitable sequences for the 5'-tail
is possible for one skilled in the art based on the requirements of
the respective application.
At the 3'-end of the anchor oligonucleotide, an additional anchor
sequence typically can be contained with a length of one to five
additional nucleotides. The anchor sequence can have a length of at
least one base, whereby the first position in a preferred
embodiment is a degenerated base, which contains all bases except
for the base that is used in the homopolymer portion of the anchor
oligonucleotide. Then, additional bases can follow. The latter can
also be degenerated. In a preferred embodiment, the use of N
wobbles is useful here, whereby N=A, C, G, T or corresponding
analogs.
The optional 5'-tail additionally comprises 1-100 nucleotides,
which can be used for subsequent analyses. Thus, in a preferred
embodiment, the 5'-tail can contain the binding sequence for an
oligonucleotide, such as, e.g., one or more DNA probes and/or one
or more PCR primers. The sequences that are used for the 5'-tail
are preferably selected such that the latter are compatible with
the process according to the invention. This comprises, e.g., the
selection of those sequences that do not cause any undesirable
secondary reactions, both in the process according to the invention
and in subsequent analytical processes.
The reaction mixture according to the invention comprises the
anchor oligonucleotide according to the invention, which has a
length of between 10 and 150 nucleotides, and optionally at the
3'-end, carries an anchor sequence according to the invention that
is one to five nucleotides long. The reaction mixture according to
the invention comprises the anchor oligonucleotide, which, as
described above, for example, is a deoxyribonucleic acid (DNA), a
peptide nucleic acid (PNA) or a locked nucleic acid (LNA). In a
preferred embodiment, the reaction mixture according to the
invention comprises an anchor oligonucleotide according to the
invention, which is a poly-(T)-oligonucleotide and in addition
carries a 5'-tail, whereby the oligonucleotide is a
deoxyribonucleic acid, which is 10 to 75 nucleotides long and is
present as a mixture, whereby on the 3'-end, an anchor sequence is
present, consisting of one nucleotide in each case, which is
selected from the group that comprises A, G and C, optionally
followed by one to five additional nucleotides that comprise all
four bases A, C, G and T or corresponding analogs.
Anchor oligonucleotides of the reaction mixture according to the
invention are shown in FIG. 12.
The reaction mixture according to the invention also comprises at
least one ribonucleotide as they were described above for the
process according to the invention. In particular, the reaction
mixture according to the invention comprises at least one
ribonucleotide that is selected from ATP, TTP, CTP, GTP, UTP or
corresponding base analogs. The ribonucleotides can optionally be
modified or labeled as described above. The reaction mixture
according to the invention comprises deoxyribonucleotides, as it
was described for the process according to the invention. In
particular, the reaction mixture according to the invention
comprises one or more deoxyribonucleotides, such as, for example,
dATP, dCTP, dGTP, dUTP, and/or dTTP. In a preferred embodiment, a
mixture of deoxyribonucleotides, which allow a cDNA synthesis, is
used. These deoxyribonucleotides can optionally be modified or
labeled.
If a deoxyribonucleotide of the reaction mixture according to the
invention is labeled, the labeling can be selected from the group
that comprises .sup.32P, .sup.33P, .sup.35S, .sup.3H, a fluorescent
dye such as, for example, fluorescein isothiocyanate (FITC),
6-carboxyfluorescein (FAM), xanthene, rhodamine,
6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX),
6-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein (JOE),
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA),
6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5),
6-carboxyrhodamine-6G (RG6), rhodamine 110; Cy3, Cy5, Cy7,
coumarins, such as umbelliferone, benzimides, such as Hoechst
33258; phenanthridines, such as Texas Red, ethidium bromides,
acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes,
polymethine dyes, cyanine dyes, such as Cy3, Cy5, BODIPY dyes,
quinoline dyes and Alexa dyes. Other labels, such as the insertion
of biotin or one or more haptens, such as, e.g., digoxigenin, which
allow a direct or indirect detection of the nucleic acid. Indirect
detection, such as, e.g., via antibodies, which in turn, e.g.,
enzymatic detection via an enzyme coupled to an antibody. Also, via
the introduction of nanoparticles, which are coupled to, e.g.,
antibodies or an affinity ligand, an indirect detection is
possible.
The reaction mixture according to the invention in each case
comprises a deoxyribonucleotide at a concentration of 0.01 mmol to
10 mmol. The individual deoxyribonucleotide A, C, G and T is
preferably present at a concentration of 0.2 mmol to 2 mmol in each
case. It is especially preferred if the deoxyribonucleotides A, C,
G and T are present together. Each individual one is present in
this preferred embodiment at a concentration of 0.5 mmol.
In addition, the reaction mixture according to the invention
comprises a buffer. This buffer has a pH of 6 to 10. In addition,
in the reaction mixture according to the invention, Mg.sup.2+ ions
are found. In an especially preferred embodiment, the reaction
mixture according to the invention has a buffer with a pH of 6.8 to
9. The reaction mixture can further comprise additional ions, which
can be selected from the group that comprises Mn.sup.2+, K+,
NH.sup.4+, and Na+. The presence of two different enzyme activities
is essential for the reaction mixture according to the invention.
The reaction mixture according to the invention comprises at least
a first enzyme activity with polyadenylation activity and secondly,
a second enzyme activity with reverse transcriptase activity. The
preferred embodiments of these activities were already described
above for the process. In addition, like the process above, the
reaction mixture can comprise additional substances, such as, for
example, a volume excluder, a single-strand binding protein, DTT,
or one or more competitor-nucleic acids.
If a volume excluder is used, it is preferred that the latter be
selected from the group that comprises dextran and polyethylene
glycol. Other volume excluders according to this invention are
found in EP1411133A 1.
If the reaction mixture optionally comprises a competitor-nucleic
acid, the latter is selected from the group that comprises
homopolymer ribonucleic acids and polyadenoribonucleic acid. Other
competitor-nucleic acids according to the invention are disclosed
in U.S. Pat. No. 6,300,069.
The reaction mixture according to the invention preferably
comprises additional poly-(C)-polynucleotides. The reaction mixture
according to the invention especially preferably comprises
additional poly-(C)-polyribonucleotides. 1 ng to 300 ng of
poly-(C)-polyribonucleotides is preferably incorporated for each 20
.mu.l, 10 ng to 150 ng of poly-(C)-polyribonucleotides is
preferably incorporated for each 20 .mu.l of reaction, 25 ng to 100
ng of poly-(C)-polyribonucleotides is especially preferably
incorporated for each reaction, and 50 ng to 75 ng of poly-(C)
polyribonucleotides is most preferably incorporated for each 20
.mu.l of reaction.
In addition, the invention relates to a kit, comprising a reaction
mixture, as it was described above. The reaction mixture is present
in a preferred embodiment in a single reaction vessel. In another
embodiment, the kit comprises a reaction vessel, comprising the
enzyme with polyadenylation activity, the enzyme with reverse
transcriptase activity, optionally the deoxyribonucleotides,
optionally at least one ribonucleotide, optionally a buffer
containing Mg.sup.2+, and optionally one or more
oligodeoxyribonucleotides in terms of the invention. Optionally,
the reaction vessel in the kit according to the invention can
contain additional components, as they were indicated for the
reaction mixture according to the invention. In addition, the kit
can comprise a probe for the 5'-tail of the anchor oligonucleotide
according to the invention. In addition, the kit can contain one or
more additional deoxyribonucleotides, thus, e.g., a generic primer
for detecting the tail sequence that is introduced by reverse
transcription. The reaction mixture can be present in "pellet
form," thus, e.g., freeze-dried. Additional preparation processes,
which do not contain, e.g., liquid forms, are known to one skilled
in the art.
In addition, the kit can optionally be combined with reagents as
they are necessary for the PCR reaction or real-time PCR reaction.
These reagents are preferred for at least one PCR reaction, which
allows the detection of at least one of the cDNAs generated in the
process according to the invention.
In addition, the kit can comprise optional random primers as well
as optionally one or more primers or primer/probes to detect
additional target genes in singleplex or multiplex PCR reactions
and/or real-time singleplex or multiplex PCR reactions.
The reaction mixture, the process according to the invention or the
kit can contain additional target-specific primers. The length of
the target-specific primer should be selected such that a specific
detection in a PCR reaction is possible; the sequence of the target
primer should be specific, such that a binding to only one spot in
the generated cDNA sequence is possible. Normally, such a primer
has a length of 15 to 30 nucleotides, preferably 17 to 25
nucleotides.
In an especially preferred embodiment, the reaction mixture
comprises the enzyme with polyadenylation activity and the enzyme
with reverse transcriptase activity as a two-enzyme pre-mix. In
this especially preferred embodiment, the kit comprises the
two-enzyme-premix-reaction mixture in a reaction vessel and in a
separate vessel, a buffer, Mg.sup.2+, rNTP(s), dNTP(s), optionally
one anchor oligonucleotide according to the invention, and
optionally random primers, as well as, in addition, optionally a
volume excluding reagent and/or a competitor-nucleic acid.
The process according to the invention can, as already explained
above, take place in one or more temperature steps. In a preferred
embodiment, the process according to the invention takes place in a
single temperature step for the incubation and another temperature
step for the inactivation of the enzyme. Thus, in a preferred
embodiment of the incubation step in which the enzyme activity
develops, it is about 37.degree. C. The incubation time is
approximately 1 to 120 minutes, preferably 5 to 90 minutes, more
preferably 10 to 75 minutes, still more preferably 15 to 60
minutes, still more preferably 20 to 60 minutes to most preferably
50 to 70 minutes. Normally, excessive incubation time is not
harmful. In another step that uses the denaturation of the enzymes,
a temperature of at least 65.degree. C. but at most 100.degree. C.
is used. Preferably, a temperature of about 80-95.degree. C. is
used. The denaturation takes place for a period of at least 1
minute, but for at most 30 minutes. In a preferred embodiment, it
is denatured for a period of 5 minutes.
The process according to the invention for generating cDNA can
subsequently comprise a polymerase chain reaction. If this is the
case, a primer that is specific to the tail that is introduced
during cDNA synthesis and/or a specific primer is preferably added
to the reaction mixture according to the invention. The reaction
mixture then also contains a thermostable DNA-polymerase in
addition.
The PCR reaction that subsequently takes place can also be a
quantitative PCR reaction. It can take place in an array, take
place in a microfluid system, take place in a capillary or else be
a real-time PCR. Other variants of the PCR are known to one skilled
in the art and are equally comprised by the process according to
the invention.
The invention also relates to a process for reverse transcription
of RNA in DNA, whereby the process comprises the following steps:
preparation of a sample that comprises RNA, addition of a first
enzyme with reverse transcriptase activity, a buffer, at least one
deoxyribonucleotide, an oligonucleotide, incubation of the agents
in one or more temperature steps, which are selected such that the
enzyme shows activity, whereby the reaction comprises additional
poly-(C)-polynucleotides.
Preferably, the enzyme with reverse transcriptase activity is HIV
Reverse Transcriptase, M-MLV Reverse Transcriptase, EAIV Reverse
Transcriptase, AMY Reverse Transcriptase, Thermus thermophilus DNA
Polymerase I, M-MLV RNAse H-Superscript, Superscript II,
Superscript Ill, Monsterscript (Epicenter), Omniscript Reverse
Transcriptase (Qiagen), Sensiscript Reverse Transcriptase (Qiagen),
ThermoScript, Thermo-X (both Invitrogen) or a mixture of two or
more enzymes with reverse transcriptase activity and
poly-(A)-polymerase from Escherichia coli. Especially preferred is
HIV Reverse Transcriptase.
The reaction preferably comprises poly-(C)-polyribonucleotides. 1
ng to 300 ng of poly-(C)-polyribonucleotides is incorporated for
each 20 .mu.l, preferably 10 ng to 150 ng of
poly-(C)-polyribonucleotides is incorporated for each 20 .mu.l of
reaction, especially preferably 25 ng to 100 ng of
poly-(C)-polyribonucleotides is incorporated for each reaction, and
most preferably, 50 ng to 75 ng of poly-(C)-polyribonucleotides is
incorporated for each 20 .mu.l of reaction.
In a preferred embodiment of the invention, the sample is a
ribonucleic acid that is selected from the group that comprises
prokaryotic ribonucleic acids, eukaryotic ribonucleic acids, viral
ribonucleic acids, ribonucleic acids whose origin is an archae
organism, microribonucleic acids (miRNA), small nucleolar
ribonucleic acids (snoRNA), messenger ribonucleic acid (mRNA),
transfer-ribonucleic acids (tRNA), non-polyadenylated ribonucleic
acids in general, as well as ribosomal ribonucleic acids (rRNA),
and moreover, a mixture of two or more of the above-mentioned
ribonucleic acids. In the sample, of course, poly-A RNA can also
already be contained.
As a template, an RNA can be used that is selected from the group
that comprises eukaryotic ribonucleic acids, mRNA, prokaryotic
ribonucleic acids, miRNA, snoRNA and rRNA. In the most preferred
embodiment of this invention, the sample comprises a ribonucleic
acid that is selected from the group that comprises miRNA and
snoRNA. Additional mixed samples from varying amounts of
ribonucleic acids of varying types accompanied by other substances
are preferred.
Based on these advantages of the process according to the
invention, the inventor could show that it is possible to prepare
and to identify miRNAs efficiently and without contamination. Small
amounts of RNA in general can be readily reverse transcribed with
the process.
The invention also relates to a kit for reverse transcription that
comprises an enzyme with reverse transcriptase activity and
poly-(C)-polynucleotides, preferably
poly-(C)-polyribonucleotides.
In one embodiment, the RNA is first polyadenylated before the
sample is reverse transcribed.
EXAMPLES
Example 1
Demonstration of the feasibility of a coupled, one-stage process of
a poly-A-reaction and reverse transcription in the same reaction
vessel; effect of various buffers on the efficiency of the
detection of a 22-mer RNA oligonucleotide.
In this experiment, the feasibility of a coupled, one-stage process
of a poly-(A)-polymerase reaction and reverse transcription in the
same reaction vessel should be demonstrated. For this purpose, the
coupled one-stage process was performed under various conditions.
This was, on the one hand, the buffer supplied with the
poly-(A)-polymerase, and, on the other hand, the buffer supplied
with the reverse transcriptase. In addition, a mixture of
poly-(A)-polymerase and reverse transcriptase buffers was tested.
As a control, the reaction was performed in a two-stage process in
a way similar to FIG. 1A.
The reactions were put together as indicated in Table 1.
TABLE-US-00002 TABLE 1 Poly-A-Reaction and Reverse Transcription
Batch 1 Batch 2 Batch 3 Batch 4 a/b a/b a/b a/b Data from the Final
Concentrations Two-Stage Process Reagents PAP Buffer RT Buffer
Mixture of the 1.) PAP 2.) RT Two Buffers Reaction Reaction 5x PAP
Buffer 1x 1x 1x 10x RT Buffer 1x 1x 1x MnCl.sub.2, 25 mmol Solution
2.5 mmol 2.5 mmol 2.5 mmol rATP, 10 mmol 1 mmol 1 mmol 1 mmol 1
mmol Poly-(A)-polymerase, 4 U 2 U 4 U 2 U 2 U/.mu.l (0.2 U/.mu.l)
(0.1 U/.mu.l) (0.2 U/.mu.l) (0.2 U/.mu.l) dNTP Mix (dA, dT, 0.5
mmol 0.5 mmol 0.5 mmol 0.5 mmol dG, dC, 5 mmol each) UniGAPdT
Primer, 1 .mu.mol 1 .mu.mol 1 .mu.mol 1 .mu.mol 10 .mu.mol RNase
Inhibitor, 10 U 10 U 10 U 10 U 10 U/.mu.l Sensiscript Reverse 1
.mu.l 1 .mu.l 1 .mu.l 1 .mu.l Transcriptase RNase-Free Water
Variable Variable Variable Variable Variable a) mleu7a 2 .times.
10E9 2 .times. 10E9 2 .times. 10E9 2 .times. 10E9 10 .mu.l of PAP
Copies Copies Copies Copies Reaction 1.) b) Neg Control (H.sub.2O
H.sub.2O H.sub.2O H.sub.2O H.sub.2O instead of mleu7a) a) Corn RNA
50 ng 50 ng 50 ng 50 ng b) Neg Control (H.sub.2O H.sub.2O H.sub.2O
H.sub.2O H.sub.2O instead of Corn RNA) Total Volume 20 .mu.l 20
.mu.l 20 .mu.l 10 .mu.l 20 .mu.l Incubation 1 Hour, 37.degree. C. 1
Hour 37.degree. C., 1 Hour, 37.degree. C. 5 Minutes, 93.degree. C.
Then Another Hour at 5 Minutes, 93.degree. C. 2.) RT Reaction PAP:
Poly A Polymerase RT: Reverse Transcription rATP: Adenosine
5'-Triphosphate
For this purpose, the reagents indicated in Table 2 were used.
TABLE-US-00003 TABLE 2 Materials for the Poly-A-Reaction and
Reverse Transcription Poly-(A)-Polymerase Ambion; Material Number
80 U: 2030 Sensiscript RT Kit Qiagen; Material Number 50rxn: 205211
Adenosine 5'-Triphosphate (rATP) Amersham; Material Number 25
.mu.mol: 27- 2056-01 RNase Inhibitor Promega; Material Number:
N2511 Uni GAP dT Primer 5'-TGG AAC GAG ACG ACG ACA GAC CAA GCT TCC
CGT TCT CAG CCT TTT TTT TTT TTT TTT TTT TTY VN-3' (SEQ ID NO: 2)
Corn RNA From 1 g of Ground Corn Husks with Qiagen RNeasy Mini Kit
(Cat. No. 74106); Plant Protocol with lOx Upscale (Maxi Shredder
and Column) mleu7a RNA Oligonucleotide 5'-VGA GGU AGU AGG UGG UAU
AGU U-3' (SEQ ID NO: 1)
In each case, reactions were conducted with templates (1a, 2a, 3a,
4a, all with a synthetic RNA oligonucleotide in a background of
corn RNA) or without templates (1b, 2b, 3b, 4b, all were added to
H.sub.20 instead of templates). The reactions without templates
were conducted as controls for the possible occurrence of
nonspecific background. Corn RNA was selected as background RNA
since the sequence of the 22-mer RNA oligonucleotide to be detected
does not occur in corn.
After the inactivation of the enzymes (see Table: 5 minutes at
93.degree. C.), 2 .mu.l each of the batches 1 alb to 4 alb was used
as templates in a real-time PCR. The preparation of the real-time
PCR was carried out in three-fold batches as indicated in Table 3
with QuantiTect SYBR Green PCR Kit (Catalog No. 204143) and the
primers indicated in Table 4.
TABLE-US-00004 TABLE 3 Components for SYBR Green Real-Time PCR
Final Concentration 2x SYBR Green PCR Master Mix 1x Hum Uni Primer,
10 .mu.mol 0.5 .mu.mol miRNA Primer let7/short, 10 .mu.mol 0.5
.mu.mol RNase-Free Water Variable PAP-/RT-Reaction 2 .mu.l Final
Reaction Volume 20 .mu.l
TABLE-US-00005 TABLE 4 Materials for the SYBR Green PCR (Table 4
discloses SEQ ID Nos: 4 and 3, respectively, in order of
appearance. QuantiTect SYBR Qiagen; Material Number: 204143 Green
PCR Kit (200) let 7short 5'-GAG GTA GTA GGT TGT ATA G-3' (Specific
miRNA (SEQ ID NO: 4) Primer) Hum Uni Primer 5'-AAC GAG ACG ACG ACA
GAC-3' (SEQ ID NO: 3)
The sequence 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO:3) that is
contained in the universal tail-primer Hum Uni Primer was described
in US2003/0186288A 1.
The PCR protocol consisted of an initial reactivation of the
HotStarTaq polymerase that is contained in the QuantiTect SYBR
Green PCR Master Mix for 15 minutes at 95.degree. C., followed by
40 cycles for 15 seconds at 94.degree. C., 30 seconds at 52.degree.
C., and 30 seconds at 72.degree. C. (see Table 5).
TABLE-US-00006 TABLE 5 PCR Protocol for SYBR Green Real-Time PCR
PCR Initial Reactivation 15 Minutes, 95.degree. C. Denaturation 15
Seconds, 94.degree. C. 40x Annealing 30 Seconds, 52.degree. C.
Extension (Data Acquisition) 30 Seconds, 72.degree. C. Melt
Curve
The acquisition of the fluorescence data was carried out during the
72.degree. C. extension step. The PCR analyses were performed with
an ABI PRISM 7700 (Applied Biosystems) in a reaction volume of 20
.mu.l.
The PCR products were then subjected to a melt curve analysis. The
latter was performed on an ABI PRISM 7000 real-time PCR
instrument.
TABLE-US-00007 TABLE 6 Results of a Real-Time PCR Analysis of the
Batches from Table 1 Ct CV Detector PAP/RT Template Ct Agent in %
SYBR 1) PAP Buffer a) mleu7a 25.64 Green 2 .times. 10{circumflex
over ( )}8 Copies + 25.22 25.50 0.95 Corn cDNA, 5 ng 25.64 b)
H.sub.2O in PAP - No Ct RT Reaction No Ct No Ct No Ct 2) RT Buffer
a) mleu7a 17.43 2 .times. 10{circumflex over ( )}8 Copies + 17.31
17.32 0.58 Corn cDNA, 5 ng 17.23 b) H.sub.2O in PAP- No Ct RT
Reaction No Ct No Ct No Ct 3) Mixture of a) mleu7a 30.41 Both
Buffers 2 .times. 10{circumflex over ( )}8 Copies + 30.23 30.28
0.36 Corn cDNA, 5 ng 30.21 b) H.sub.2O in PAP- No Ct RT Reaction No
Ct No Ct No Ct 4) Two-Stage a) mleu7a 25.54 Process 2 .times.
10{circumflex over ( )}8 Copies + 25.74 25.64 0.39 Corn DNA, 5 ng
25.65 b) H.sub.2O in PAP- No Ct RT Reaction No Ct No Ct No Ct
The identity of the PCR products was then examined with the aid of
agarose-gel electrophoresis. For this purpose, 10 .mu.l of each PCR
reaction was loaded onto a 2% agarose gel colored with ethidium
bromide and separated. 100 bp Lader (Invitrogen,
Catalog No. 15628-050) was used as a size standard. The results are
depicted in FIG. 3.
Example 2
Demonstration of the reproducibility and specificity of a coupled
one-stage process of poly-(A)-polymerase reaction and reverse
transcription in the same reaction vessel
In this experiment, the feasibility of a coupled one-stage process
of poly-(A)-polymerase reaction and reverse transcription should be
reproduced in the same reaction vessel. For this purpose, the
efficiency of the one-stage process of the poly-(A)-polymerase
reaction and reverse transcription in the same reaction vessel was
analyzed in the example of the detection of a 22-mer RNA
oligonucleotide. A reverse transcription reaction for the template
that is used according to standard conditions was used as a control
for the specificity of the detection.
For this purpose, the coupled one-stage process was performed under
various conditions. The latter were, on the one hand, the buffer
supplied with poly-(A)-polymerase, and, on the other hand, the
buffer supplied with the reverse transcriptase (Tables 7, 8).
TABLE-US-00008 TABLE 7 Designation and Components of the Reactions
Conducted Designation Buffer Template Reaction 1 1-Step Process RNA
50 ng + mleu7a 2 .times. 10E9 Copies Reaction 2 in PAP Buffer RNA,
50 ng Reaction 3 Negative Control: H.sub.20 Reaction 4 1-Step
Process RNA 50 ng + mleu7a 2 .times. 10E9 Copies Reaction 5 in RT
Buffer RNA, 50 ng Reaction 6 Negative Control: H20 Reaction 7
Standard RT RNa 50 ng + mleu7a 2 .times. 10E9 Copies Reaction 8
RNA, 50 ng
TABLE-US-00009 TABLE 8 Table 8: Composition of the Combined
Poly-(A)-Polymerase/Reverse Transcription Reaction and the Standard
Reverse Transcription Reaction Composition of the Combined
Poly-(A)-Polymerase/Reverse Transcription Reaction and the Standard
Reverse Transcription Reaction Data from the Final Concentrations
Reactions Reactions Reactions 1, 2, 3 4, 5, 6 7, 8 Reagents PAP
Buffer RT Buffer Standard RT 5x PAP Buffer 1x 10x RT Buffer 1x 1x
MnCl.sub.2, 25 mmol 2.5 mmol rATP, 10 mmol 1 mmol 1 mmol
Poly-(A)-Polymerase, 2 U/.mu.l 2 U 2 U (0.1 U/.mu.l) (0.1 U/.mu.l)
dNTP Mix (dA, dT, dG, dC, 0.5 mmol 0.5 mmol 0.5 mmol 5 mmol each)
UniGAPdT Primer, 10 .mu.mol 1 .mu.mol 1 .mu.mol 1 .mu.mol RNase
Inhibitor, 10 U/.mu.l 10 U 10 U 10 U Sensiscript Reverse 1 .mu.l 1
.mu.l 1 .mu.l Transcriptase RNase-Free Water Variable Variable
Variable mleu7a; Reactions 1, 4, 7 2 .times. 10E9 2 .times. 10E9 2
.times. 10E9 Copies Copies Copies RNA: Reactions 1, 2, 4, 5, 50 ng
50 ng 50 ng 7, 8 Neg. Control (H.sub.2O instead of H.sub.2O
H.sub.2O RNA) in Reactions 3, 6 Total Volume 20 .mu.l 20 .mu.l 20
.mu.l Incubation 1 Hour, 37.degree. C. 5 Minutes, 93.degree. C. All
reaction batches were then divided: Batches a) 10 .mu.l was removed
and stored at 4.degree. C.; For all batches b): Uni GAP dT primer
[1 .mu.mol] and 0.5 .mu.l of Sensiscript Reverse Transcriptase were
added again to 10 .mu.l, and a reverse transcription was performed
again (for 1 hour at 37.degree. C.), then the reverse transcriptase
was inactivated (5 minutes, 93.degree. C.).
In addition, a standard reverse transcription reaction was
performed with the purpose of examining the specificity of the
detection in the subsequent PCR (Tables 7, 8, see above). After the
poly-(A)-polymerase reaction and reverse transcription, the samples
were divided. In each case, Uni Gap dT primer and reverse
transcriptase were added again to half of a sample (see Table 7,
above). The purpose here was to rule out the possibility that
false-positive signals are produced to a small extent by an
undesired adherence of an A-Tail to the Uni Gap dT primer. As a
template, total-RNA was added, which was isolated from human blood
with an RNeasy Midi Kit (QIAGEN, Hilden, Germany, Cat. No.
75144).
The components that are used in the individual reactions and their
designations are put together in Table 7, see above. The detected
22-mer RNA corresponds in the sequence thereof to the human leu7a
miRNA (EMBL Ace #: AJ421724) and may be expressed in human blood
cells such as leukocytes. However, such small RNAs are only very
inefficiently purified because of the purification technology of
the RNeasy process that is used for the RNA isolation. The RNeasy
process ensures only an efficient binding of RNAs with a size above
200 bases on the silica membrane of the RNeasy column (QIAGEN
RNeasy Midi/Maxi Handbook, June 2001, p. 9), and thus small RNAs
such as miRNAs are stripped out to a large extent.
In addition, the synthetic 22-mer RNA was used at a concentration
that is clearly higher than the endogenic copy number expected. The
reactions were put together as indicated in Table 8 (see
above).
To this end, the reagents indicated in Table 9 were used.
TABLE-US-00010 TABLE 9 Materials for the Poly-A-Reaction and
Reverse Transcription Poly (A) Polymerase Ambion; Material Number
80 U: 2030 Sensiscript RT Kit Qiagen; Material Number 50rxn: 205211
Adenosine 5'-Triphosphate (rATP) Amersham; Material Number 25
.mu.mol: 27-2056-01 RNase Inhibitor Promega; Material Number: N2511
Uni GAP dT Primer 5'-TGG AAC GAG ACG ACG ACA GAC CAA GCT TCC CGT
TCT CAG CCT TTT TTT TTT TTT TTT TTT TTY V(N-Q)-3' (SEQ ID NO: 2)
RNA RNA Leukocytes from Human Blood Isolated with an RNeasy Midi
Kit (QIAGEN, Cat. No. 75144) mleu7a RNA Oligonucleotide 5'-UGA GGU
AGU AGG UUG UAU AGU U-3' (SEQ ID NO: 1)
After the inactivation of the enzymes (5 minutes at 93.degree. C.),
the batches were diluted 1:2 with water, and 2 .mu.l each of the
batches 1a/b to 8a/b was used as a template in a real-time SYBR
Green PCR. The preparation of the real-time PCR was carried out in
two-fold batches as indicated in Table 9 (above) with QuantiTect
SYBR Green PCR Kit (Catalog No. 204143) and the primers indicated
in Table 10.
TABLE-US-00011 TABLE 10 Components for SYBR Green Real-Time PCR
Final Concentration 2x SYBR Green PCR Master Mix 1x Hum Uni Primer,
10 .mu.mol 0.5 .mu.mol miRNA Primer let 7short, 10, .mu.mol 0.5
.mu.mol RNase-Free Water Variable PAP-/RT-Reaction 1:2 prediluted 2
.mu.l Final Reaction Volume 20 .mu.l
The sequence 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO: 3) contained
in the universal tail-primer Hum Uni Primer was described in US
2003/0186288A 1. The PCR protocol consisted of an initial
reactivation of the HotStarTaq Polymerase contained in the
QuantiTect SYBR Green PCR Master Mix for 15 minutes at 95.degree.
C., followed by 40 cycles for 15 seconds at 94.degree. C., 30
seconds at 52.degree. C., and 30 seconds at 72.degree. C. (see
Table 11).
TABLE-US-00012 TABLE 11 Materials for the SYBR Green PCR QuantiTect
SYBR Qiagen; Material Number: 204143 Green PCR Kit (200) let 7short
5'-GAG GTA GTA GGT TGT ATA G-3' (Specific miRNA (SEQ ID NO: 4)
Primer) Hum Uni Primer 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO:
3)
The acquisition of the fluorescence data was carried out during the
72.degree. C. extension step. The PCR analyses were performed with
an Applied Biosystems 7500 Fast Real-Time PCR System (Applied
Biosystems) in a reaction volume of 20 .mu.l and then a melt curve
analysis was performed.
The coupled one-stage poly-(A)-reaction and reverse transcription
are possible both in poly-(A)-polymerase buffer and in RT buffer,
which is evident based on real-time PCR analyses. Preferred buffer
conditions were already indicated in the text (see above). They
show large differences in the Ct values that are obtained when
using the different buffers.
The standard reverse transcription reactions (reactions 3, 6),
performed for the monitoring of the specificity, are all negative
(no Ct) without poly-(A)-reactions. This allows the conclusion that
without polyadenylation of the 22-mer RNA (1, 4, 7) or the
naturally occurring miRNA (2, 5, 8) as expected, no template for a
PCR amplification is present and therefore no signal can be
generated ("no Ct").
In the "RT doubled" batches, additional RT enzymes and Uni Gap dT
Primer were added after the first incubation with the purpose of
making poly-(A)-tailed UniGap dT Primer detectable by an RT
reaction, possibly in the first reaction. All of these batches
showed no Ct, i.e., undesirable artifacts are not detectable.
Undesirable artifacts such as poly-(A)-tailing of the primer used
for the cDNA synthesis are also not detectable.
Example 3
Detection of various miRNAs using the process according to the
invention.
In this experiment, it should be demonstrated that several targets
can be detected by miRNA-specific PCR Primers from a cDNA template
that was synthesized with the process according to the invention
with poly-(A)-reactions of common reverse transcription. For this
purpose, a process was performed with 293 RNA as a template. A
reverse transcription reaction for the template that is used
according to standard conditions was used as a control for the
specificity of the detection.
In the subsequent SYBR Green PCR, overall in each case one of 4
different specific primers for miRNAs together with the tail of
specific primers was used. In addition, a primer located on the
3'-end of the human B-actin transcript was used together with a
tail-specific primer to examine the efficiency of the
poly-A-reaction and reverse transcription.
For the poly-A-reaction and reverse transcription (PAP+RT
reaction), the reagents from Table 13 were pipetted together as
indicated in Table 16.
TABLE-US-00013 TABLE 13 Materials for the Poly A Reaction and
Reverse Transcription Poly (A) Polymerase Ambion; Material Number
80 U: 2030 Sensiscript RT Kit Qiagen; Material Number 50rxn: 205211
Adenosine 5'- Amersham; Material Number 25 .mu.mol: 27-
Triphosphate (rATP) 2056-01 RNase Inhibitor Promega; Material
Number: N2511 Uni GAP dT Primer 5'-TGG AAC GAG ACG ACG ACA GAC CAA
GCT TCC CGT TCT CAG CCT TTT TTT TTT TTT TTT TTT TTY VN-3' (SEQ ID
NO: 2) 293 RNA: From Human Cell Line 293 (ATCC Number: CRL-1573)
With Qiagen RNeasy Midi Kit Isolated
TABLE-US-00014 TABLE 16 PAP + RT Reaction Final Reagents
Concentration 1Ox Buffer RT 1x rATP, 10 mmol 1 mmol Poly A
Polymerase 2 U/.mu.l 2 U dNTP Mix (ATGC, 5 mmol each) 0.5 mmol Uni
GAP dT Primer, 10 .mu.mol 1 .mu.mol RNase Inhibitor, 10 U/.mu.l 10
U Sensiscript Reverse Transcriptase 1 .mu.l RNase-Free Water
Variable a) 293 RNA, 20 ng/.mu.l 5 .mu.l (100 ng) b) H.sub.20 for
Neg Control 5 .mu.l Total Volume 20 .mu.l Incubation 1 Hour,
37.degree. C. 5 Minutes, 93.degree. C.
In reaction a), 293 RNA was added as a negative control (neg
control) and in reaction b), water was added as a negative control.
As a control for the specificity of the detection, a standard
reverse transcription reaction with the reagents indicated in Table
14 was prepared based on the diagram in Table 17.
TABLE-US-00015 TABLE 14 Materials for Reverse Transcription
Sensiscript Qiagen; Material Number 50rxn: 205211 RT Kit RNase
Promega; Material Number: N2511 Inhibitor Uni GAP 5'-TGG AAC GAG
ACG ACG ACA GAC dT Primer CAA GCT TCC CGT TCT CAG CCT TTT TTT TTT
TTT TTT TTT TTY VN-3' (SEQ ID NO: 2) 293 RNA With Qiagen RNeasy
Midi Kit Isolated
TABLE-US-00016 TABLE 17 Standard RT Reaction Reagents 2.) RT
Reaction 1Ox Buffer RT 1x dNTP Mix (ATGC, 5 mmol each) 0.5 mmol Uni
GAP dT Primer, 10 .mu.mol 1 .mu.mol RNase Inhibitor, 10 U/.mu.l 10
U Sensiscript Reverse Transcriptase 1 .mu.l RNase-Free Water
Variable c) 293 RNA, 20 ng/.mu.l 5 .mu.l (100 ng) d) H.sub.20 for
Neg Control 5 .mu.l Total Volume 20 .mu.l Incubation 1 Hour,
37.degree. C. 5 Minutes, 93.degree. C.
In reaction c), 293 RNA was added as negative control (neg
control), and in reaction d), water was added as negative control.
The samples were then incubated for one hour at 37.degree. C. To
stop the reaction, the reactions were incubated for 5 minutes at
93.degree. C.; the enzymes are inactivated by this temperature
step.
After the inactivation of the enzymes, the batches 1:2 were diluted
with water and 2 .mu.l each of the batches a) to d) were used as
templates in a real-time SYBR Green PCR. The materials for the PCR
are indicated in Table 15.
TABLE-US-00017 TABLE 15 Materials for the SYBR Green PCR QuantiTect
SYBR Green PCR Kit (200) Qiagen; Material Number: 204143 let 7short
(Specific miRNA Primer) 5'-GAG GTA GTA GGT TGT ATA G-3' (SEQ ID NO:
4) hsa-miR-24 (Specific miRNA Primer) 5'-TGG CTC AGT TCA GCA GGA-3'
(SEQ ID NO: 5) hsa-miR-15a (Specific miRNA Primer) 5'-TAG CAG CAC
ATA ATG GTT T-3' (SEQ ID NO: 6) hsa-miR-16 (Specific miRNA Primer)
5'-TAG CAG CAC GTA AAT ATT G-3' (SEQ ID NO: 7) B-Actin 3' Primer
5'-GTA CAC TGA CTT GAG ACC AGT TGA ATA AA-3' (SEQ ID NO: 8) Hum Uni
Primer 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO: 3)
Ten different reaction batches were pipetted. In reactions 1-5
(Table 18), in each case the miRNA-specific or B-actin 3' primer
and the tail primer (Hum Uni) were used.
TABLE-US-00018 TABLE 18 SYBR GREEN PCR Reactions 1-5 Final
Components for SYBR Green PCR Concentration 2x SYBR Green PCR
Master Mix 1x Hum Uni Primer, 10 .mu.mol 0.5 .mu.mol One Specific
miRNA Primer Each (10 .mu.mol) 0.5 .mu.mol let 7short hsa-miR-24
hsa-miR-15a hsa-miR-16 RNase-Free Water Variable PAP + RT Reaction
a) b) 1:2 prediluted 2 .mu.l (5 ng) Standard RT Reaction c) d) 1:2
prediluted 2 .mu.l (5 ng) or H.sub.20 as Neg Control 2 .mu.l Final
Reaction Volume 20 .mu.l
In reactions 6-10 (Table 19), in each case only one primer was
used, either the miRNA specific primer or the tail-specific
primer.
In reactions 6-10 (Table 19), in each case only one primer was
used, either the miRNA specific primer or the tail-specific
primer.
TABLE-US-00019 TABLE 19 Control with Only One Primer Reaction 6-10
Final Components for SYBR Green PCR Concentration 2x SYBR Green PCR
Master Mix 1x One Specific miRNA Primer Each (10 .mu.mol) 0.5
.mu.mol let 7short hsa-miR-24 hsa-miR-15a hsa-miR-16 Each with 3'
Hum Uni Primer RNase-Free Water Variable PAP + RT Reaction a) b)
1:2 Prediluted 2 .mu.l (5 ng) Standard RT Reaction c) d) 1:2
Prediluted 2 .mu.l (5 ng) or H.sub.20 as Neg Control 2 .mu.l Final
Reaction Volume 20 .mu.l
The sequence in which the primers were used in the reactions can be
seen from Table 20. The preparation of the real-time PCR was
carried out in two-fold batches.
TABLE-US-00020 TABLE 20 Primer Primer Reaction 1 .beta.-Actin
3'Primer + Hum Uni Reaction 2 let 7short + Hum Uni Reaction 3
hsa-miR-24 + Hum Uni Reaction 4 hsa-miR-15a + Hum Uni Reaction 5
hsa-miR-16 + Hum Uni Reaction 6 Hum Uni Reaction 7 let 7short
Reaction 8 hsa-miR-24 Reaction 9 hsa-miR-15a Reaction 10
hsa-miR-16
The sequence AAC GAG ACG ACG ACA GAC (SEQ ID NO: 3) contained in
the universal Tail-Primer Hum Uni Primer was described in
US2003/0186288A 1.
The PCR protocol consisted of an initial reactivation of the
HotStarTaq polymerase contained in the QuantiTect SYBR Green PCR
Master Mix for 15 minutes at 95.degree. C., followed by 40 cycles
for 15 seconds at 94.degree. C., 30 seconds at 52.degree. C., and
30 seconds at 72.degree. C. (see Table 21). The acquisition of the
fluorescence data was carried out during the 72.degree. C.
extension step. The PCR analyses were performed with an Applied
Biosystems 7000 Fast Real-Time PCR System (Applied Biosystems) in a
reaction volume of 20 .mu.l, and then a melt curve analysis was
performed.
TABLE-US-00021 TABLE 21 3-Step PCR Protocol PCR Initial
Reactivation 15 Minutes, 95.degree. C. Denaturation 15 Seconds,
94.degree. C. 40x Annealing 30 Seconds, 52.degree. C. Extension 30
Seconds, 72.degree. C. Melt Curve
It is shown that the efficiency of the reverse transcription
performed under standard conditions and of the process according to
the invention for the B-actin system that is selected by way of
example is comparable, which is evident in comparable Ct values in
the real-time PCR (FIG. 6).
When using the cDNA produced under standard conditions, the
real-time PCR yields very high Ct values of above 38, which mean a
very good specificity of the detection in a real-time PCR that is
performed with SybrGreen (FIG. 6). In the agarose gel analysis of
the PCR products, PCR products of the expected values were detected
(FIG. 8), or no product was detected when using the cDNA produced
under standard conditions.
The miR24 product represents an acquisition. Here, when the cDNA
produced under standard conditions is used, a PCR product of the
wrong size is produced (FIG. 8, see also FIG. 6). This product
cannot be detected, as soon as a cDNA is used, which was produced
using the process according to the invention (FIG. 8).
All control reactions in which water instead of RNA template was
used in the reverse transcription or the process according to the
invention show no signal, i.e., no Ct value in the real-time PCRs
in question was obtained (FIG. 7, upper part). The same also
applies for reactions in which only one primer was used (FIG. 7,
upper part). Also, no Ct value was obtained for negative controls,
in which water instead of cDNA was used in the PCR (FIG. 7).
Example 4
Detection of miRNA using the coupled, one-stage process of
poly-A-reaction and reverse transcription and subsequent detection
of the generated cDNA over real-time PCR with a tail-specific
probe.
In this experiment, a real-time PCR was performed, in which a
Taqman probe was used, which has a specific binding site on the
tail-primer (Uni Gap dT). The detection via a probe represents a
conceivable alternative for the detection via SYBR Green real-time
PCR. The use of the probe offers the additional possibility of a
multiplex PCR, i.e., a co-amplification or one or more additional
target nucleic acids, such as an internal control, which can be,
e.g., a housekeeping gene.
For the poly-A-reaction and reverse transcription (PAP+RT
reaction), the reagents from Table 22 were pipetted together as
indicated in Table 24.
TABLE-US-00022 TABLE 22 Materials for the Poly A Reaction and
Reverse Transcription Poly (A) Polymerase Ambion; Material Number
80 U: 2030 Sensiscript RT Kit Qiagen; Material Number 50rxn: 205211
Adenosine Amersham; Material Number 25 .mu.mol: 27- 5'-Triphosphate
(rATP) 2056-01 RNase Inhibitor Promega; Material Number; N2511 Uni
GAP dT Primer 5'-TGG AAC GAG ACG ACG ACA GAC CAA GCT TCC CGT TCT
CAG CCT TTT TTT TTT TTT TTT TTT TTY VN-3' (SEQ ID NO: 2) mleu7a
Oligonucleotide 5'-VGA GGU AGU AGG UUG UAU AGU U-3' (SEQ ID NO:
1)
TABLE-US-00023 TABLE 24 PAP + RT Reaction Final Reagents
Concentration 10x Buffer RT 1x rATP, 10 mmol 1 mmol Poly A
Polymerase, 2 U/.mu.l 2 U dNTP Mix (ATGC, 5 mmol each) 0.5 mmol Uni
GAP dT Primer, 10 .mu.mol 1 .mu.mol RNase Inhibitor, 10 U/.mu.l 10
U Sensiscript Reverse Transcriptase 1 .mu.l RNase-Free Water
Variable mleu7 (10''9 Copies/.mu.l) 2 .mu.l (2 .times. 10''9
Copies) Total Volume 20 .mu.l Incubation 1 Hour, 37.degree. C. 5
Minutes, 93.degree. C.
Then, the reaction batch was incubated at 37.degree. C., followed
by an inactivation of the enzymes for 5 minutes to 93.degree.
C.
After the inactivation of the enzymes, 2 .mu.l of the undiluted
batch was used as a template in a real-time PCR, which contained a
Taqman probe for detection. The materials for the PCR are indicated
in Table 23 and were pipetted together as indicated in Table
25.
TABLE-US-00024 TABLE 23 Materials for the QT Probe PCR QuantiTect
Probe Qiagn Material No.: 204343 PCR Kit (200) let 7short 5'-GAG
GTA GTA GGT TGT ATA G-3' (Specific miRNA (SEQ ID NO: 4) primer) Hum
Uni Primer 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO: 3) Hum Uni
Probe 5'-HEX-CAA GCT TCC CGT TCT CAG CC-BHQ-3' (SEQ ID NO: 9) 5'
Reporter Dye: HEX 3' Quencher: Black Hole Quencher 1
TABLE-US-00025 TABLE 25 QuantiTect Probe PCR Final Components for
QuantiTect Probe PCR Concentration 2x QuantiTect Probe PCR Master
Mix 1x Hum Uni Primer, 10 .mu.mol 0.5 .mu.mol let 7short (specific
miRNA Primer) 0.5 .mu.mol RNase-Free Water Variable PAP + RT
Reaction, Undiluted 2 .mu.l (2 .times. 10''8 Copies) Final Reaction
Volume 20 .mu.l
The batch of real-time PCR was carried out in two-fold batches.
The sequence AAC GAG ACG ACG ACA GAC (SEQ ID NO: 3) contained in
the universal tail-primer Hum Uni Primer was described in
US2003/0186288A 1. The Taqman probe sequence was removed in the
human GAPDH gene locus; it is not contained in US2003/0186288A
1.
The PCR protocol consisted of an initial reactivation of the
HotStarTaq polymerase contained in the QuantiTect Probe PCR Master
Mix for 15 minutes at 95.degree. C., followed by 45 cycles for 15
seconds at 94.degree. C. and 30 seconds at 52.degree. C. (see Table
26).
TABLE-US-00026 TABLE 26 2-Step PCR Protocol PCR Initial
Reactivation 15 Minutes, 95.degree. C. Denaturation 15 Seconds,
94.degree. C. 45x Annealing 30 Seconds, 52.degree. C.
The acquisition of the fluorescence data was carried out during the
52.degree. C. annealing step. The PCR analyses were performed with
a 7700 sequence detection system (Applied Biosystems) in a reaction
volume of 20 .mu.l. The PCR results are shown in Table 27.
TABLE-US-00027 TABLE 27 PCR Results mleu7a Ct Ct Agent CV in %
Undiluted, 2 .times. 10''8 20.24 20.31 0.45 20.37
A detection using a tail-specific probe is possible and yields the
expected result.
Example 5
Effect of poly-A-polymerase concentration and incubation time in
the coupled, one-stage process of poly-A-reaction and reverse
transcription.
In this test, two concentrations of poly-A-polymerase (2 U or 0.5
U) were used for 15 minutes or 1 hour respectively in the process
according to the invention. All conditions were tested in each case
in the RT buffer (Qiagen) and poly-A-polymerase buffer.
For the poly-A-reaction and reverse transcription (PAP+RT
reaction), the reagents from Table 28 were pipetted together as
indicated in Table 30: reaction a) 2 U of poly-A-polymerase,
reaction b) 0.5 U of poly-A-polymerase).
TABLE-US-00028 TABLE 28 Materials for the Poly A Reaction and
Reverse Transcription Poly (A) Polymerase Ambion; Material Number
80 U: 2030 Sensiscript RT Kit Qiagen; Material Number 50rxn: 205211
Adenosine Amersham; Material Number, 25 .mu.mol: 27-
5'-Triphosphate (rATP) 2056-01 RNase Inhibitor Promega; Material
Number: N2511 Uni GAP dT Primer 5'-TGG AAC GAG ACG ACG ACA GAC CAA
GCT TCC CGT TCT CAG CCT TTT TTT TTT TTT TTT TTT TTY VN-3' (SEQ ID
NO: 2) Corn RNA From 1 g of Ground Corn Husks with Qiagen RNeasy
Mini Kit (Cat. No. 74106) according to Plant Protocol. mleu7a
Oligonucleotide 5'-VGA GGU AGU AGG UUG UAU AGU U-3' (SEQ ID NO:
1)
TABLE-US-00029 TABLE 30 PAP + RT Reaction Final Reagents
Concentration 10x Buffer RT 1x rATP, 10 mmol 1 mmol Poly A
Polymerase, 2 U/.mu.l a) 2 U b) 0.5 U dNTP Mix (ATGC, 5 mmol each)
0.5 mmol Uni GAP dT Primer, 10 .mu.mol 1 .mu.mol RNase Inhibitor,
10 U/.mu.l 10 U Sensiscript Reverse Transcriptase 1 .mu.l
RNase-Free Water Variable mleu7 a 10''9 Copies/.mu.l 2 .mu.l (2
.times. 10''9 Copies) Corn RNA, 25 ng/.mu.l 2 .mu.l (50 ng) Total
Volume 20 .mu.l 1.) Incubation 1 Hour, 37.degree. C. 5 Minutes,
93.degree. C. 2.) Incubation 15 Minutes, 37.degree. C. 5 Minutes,
93.degree. C.
Then, the samples were incubated at 37.degree. C. (1. 1 hour/2. 15
minutes). Then, the reactions were heated for 5 minutes to
93.degree. C., and thus the enzymes were inactivated.
Then, in each case 2 .mu.l was incorporated undiluted into an SYBR
Green PCR for each reaction. The reactions were tested two times
apiece. For this purpose, the reagents from Table 29 were pipetted
together as indicated in Table 31, and then the PCR was performed
as indicated in Table 32.
TABLE-US-00030 TABLE 29 Materials for the SYBR Green PCR QuantiTect
SYBR Qiagen; Material Number: 204143 Green PCR Kit (200) let 7short
5'-GAG GTA GTA GGT TGT ATA G-3' (Specific miRNA (SEQ ID NO: 4)
Primer) Hum Uni Primer 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO:
3)
TABLE-US-00031 TABLE 31 SYBR GREEN PCR Final Components for SYBR
Green PCR Concentration 2x SYBR Green PCR Master Mix 1x Hum Uni
Primer, 10 .mu.mol 0.5 .mu.mol let 7short (Specific miRNA Primer)
0.5 .mu.mol RNase-Free Water Variable PAP + RT Reaction 1a) b)/2a)
b) 2 .mu.l (2 .times. 10''8 Copies) Final Reaction Volume 20
.mu.l
TABLE-US-00032 TABLE 32 3-Step PCR Protocol PCR Initial
Reactivation 15 Minutes, 95.degree. C. Denaturation 15 Seconds,
94.degree. C. 45x Annealing 30 Seconds, 52.degree. C. Extension 30
Seconds, 70.degree. C. Melt Curve
The PCR protocol consisted of an initial reactivation of the
HotStarTaq polymerase contained in the QuantiTect SYBR Green PCR
Master Mix for 15 minutes at 95.degree. C., followed by 40 cycles
for 15 seconds at 94.degree. C., 30 seconds at 52.degree. C., and
30 seconds at 72.degree. C. (see Table 32 above). The acquisition
of the fluorescence data was carried out during the 72.degree. C.
extension step. The PCR analyses were performed with an Applied
Biosystems 7000 Real-Time PCR System (Applied Biosystems) in a
reaction volume of 20 .mu.l, and then a melt curve analysis was
performed.
A dependency of the efficiency of the one-stage process of the
poly-A-reaction and reverse transcription both on the concentration
of the poly-A-polymerase and on the incubation time can be seen
(see FIG. 9).
Example 6
Implementation of the process according to the invention with
various reverse transcriptases.
The process according to the invention was applied with a total of
five different reverse transcriptases (see Table 35) in the buffer
RT (Qiagen) (reactions 1-5) and additionally, for purposes of
comparison, in each case in the buffer supplied with the reverse
transcriptase (reactions 6-9).
TABLE-US-00033 TABLE 35 Reverse Transcriptases and Buffers that are
Used 1.) AMV Reverse Transcriptase AMV Reverse Transcriptase 5 x
2.) SuperScript III Reverse Reaction Buffer Transcriptase 5 x
First-Strand Buffer 3.) HIV Reverse Transcriptase 10 x First-Strand
Synthesis Buffer 4.) M-MuLV Reverse Transcriptase 10 x Reverse
Transcriptase 5.) Sensiscript Reverse Reaction Buffer Transcriptase
10 x Buffer RT
For the one-stage process of poly-A-reaction and reverse
transcription (PAP+RT reaction), the reagents from Table 33 were
pipetted together for reactions 1-5 (reaction buffer: buffer RT
(Qiagen) as indicated in Table 36 and for reactions 6-9 (additional
reverse transcriptases, in each case in the buffer that is
supplied) as indicated in Table 37.
TABLE-US-00034 TABLE 33 Materials for the Poly A Reaction and
Reverse Transcription Poly (A) Polymerase Ambion; Material Number
80 U: 2030 AMY Reverse Transcriptase Promega; Material Number 300
U: M5101 SuperScript III Reverse Transcriptase Invitrogen; Material
Number 10,000 U: 18080-044 HIV Reverse Transcriptase Ambion;
Material Number 500 U: #2045 M-MuLV Reverse Transcriptase BioLabs;
Material Number 10,000 U: M0253S Sensiscript RT Kit Qiagen;
Material Number 50 rxns: 205211 Adenosine 5'-Triphosphate (rATP)
Amersham; Material Number 25 .mu.mol: 27- 2056-01 RNase Inhibitor
Promega; Material Number: N2511 Uni GAP dT Primer 5'-TGG AAC GAG
ACG ACG ACA GAC CAA GCT TCC CGT TCT CAG CCT TTT TTT TTT TTT TTT TTT
TTY V-3' (SEQ ID NO: 2) Corn RNA From 1 g of Ground Corn Husks with
Qiagen RNeasy Mini Kit (Cat. No. 74106) Plant Protocol, 10-Fold
Upscale (Maxi Shredder and Column) mleu7a Oligonucleotide 5'-VGA
GGU AGU AGG UUG UAU AGU U-3' (SEQ ID NO: 1)
TABLE-US-00035 TABLE 36 PAP + RT Reaction in Buffer RT (Qiagen)
Final Reagents Concentration 10x Buffer RT 1x rATP, 10 mmol 1 mmol
Poly A Polymerase, 2 U/.mu.l 1 U dNTP Mix (ATGC, 5 mmol each) 0.5
mmol Uni GAP dT Primer, 10 .mu.mol 1 .mu.mol RNase Inhibitor 10
U/.mu.l 10 U 1.) AMV Reverse Transcriptase 24 U 2.) SuperScript III
Reverse Transcriptase 10 U 3.) HIV Reverse Transcriptase 1 U 4.)
M-MuLV Reverse Transcriptase 10 U 5.) Sensiscript Reverse
Transcriptase 1 .mu.l RNase-Free Water Variable Mleu7 a 10''9
Copies/.mu.l 2 .mu.l (2 .times. 10''9 Copies) Corn RNA, 20 ng/.mu.l
2 .mu.l (40 ng) Total Volume 20 .mu.l Incubation 1 Hour, 37.degree.
C. 5 Minutes, 93.degree. C.
TABLE-US-00036 TABLE 37 PAP + RT Reaction in Buffer Supplied with
the Reverse Transcriptase Final Reagents Concentration 6.) AMV
Reverse Transcriptase 5 x Reaction Buffer 1x 7.) 5 x First-Strand
Buffer 1x 8.) 10 x First-Strand Synthesis Buffer 1x 9.) 10 x
Reverse Transcriptase Reaction Buffer 1x rATP, 10 mmol 1 mmol Poly
A Polymerase, 2 U/.mu.l 1 U dNTP Mix (ATGC, 5 mmol each) 0.5 mmol
Uni GAP dT Primer, 10 .mu.mol 1 .mu.mol RNase Inhibitor, 10 U/.mu.l
10 U 6.) AMV Reverse Transcriptase 24 u 7.) 7.) SuperScript III
Reverse Transcriptase 10 U 8.) HIV Reverse Transcriptase 1 U 9.)
M-MuLV Reverse Transcriptase 10 U RNase-Free Water Variable mleu7 a
10''9 Copies/.mu.l 2 .mu.l (2 .times. 10''9 Copies) Corn RNA, 20
ng/.mu.l 2 .mu.l (40 ng) Total Volume 20 .mu.l Incubation 1 Hour,
37.degree. C. 5 Minutes, 93.degree. C.
Then, the samples were incubated for 1 hour at 37.degree. C. Then,
the reactions were heated for 5 minutes to 93.degree. C., and thus
the enzymes were inactivated.
Subsequently, in each case 2 .mu.l was incorporated into an SYBR
Green PCR for each reaction. The reactions were tested two times
apiece. For this purpose, the reagents from Table 34 were pipetted
together as indicated in Table 38, and the PCR was performed as
indicated in Table 39.
TABLE-US-00037 TABLE 34 Materials for the SYBR Green PCR QuantiTect
SYBR Qiagen; Material Number: 204143 Green PCR Kit (200) Let 7short
5'-GAG GTA GTA GGT TGT ATA (Specific miRNA G-3' (SEQ ID NO: 4)
Primer) Hum Uni Primer 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO:
3)
TABLE-US-00038 TABLE 38 SYBR GREEN PCR Final Components for SYBR
Green PCR Concentration 2x SYBR Green PCR Master Mix 1x Hum Uni
Primer, 10 .mu.mol 0.5 .mu.mol let 7short (Specific miRNA Primer)
0.5 .mu.mol RNase-Free Water Variable PAP + RT Reaction, Undiluted
2 .mu.l (2 .times. 10{circumflex over ( )}8 Copies) Final Reaction
Volume 20 .mu.l
TABLE-US-00039 TABLE 39 3-Step PCR Protocol PCR Initial
Reactivation 15 Minutes, 95.degree. C. Denaturation 15 Seconds,
94.degree. C. 45x Annealing 30 Seconds, 52.degree. C. Extension 30
Seconds, 70.degree. C. Melt Curve
The PCR protocol consisted of an initial reactivation of the
HotStarTaq polymerase contained in the QuantiTect SYBR Green PCR
Master Mix for 15 minutes at 95.degree. C., followed by 40 cycles
for 15 seconds at 94.degree. C., 30 seconds at 52.degree. C., and
30 seconds at 72.degree. C. (see Table 39). The acquisition of the
fluorescence data was carried out during the 72.degree. C.
extension step. The PCR analyses were performed with an Applied
Biosystems 7000 Real-Time PCR System (Applied Biosystems) in a
reaction volume of 20 .mu.l, and then a melt curve analysis was
performed.
The amounts of reverse transcriptases used were optimized for
standard reverse transcription reactions, which can be a likely
explanation for the differences in the Ct value that are
observed.
Example 7
Demonstration of the feasibility of a coupled, three-stage process
of poly-A-reaction, reverse transcription and PCR in the same
reaction vessel; effect of various additions to the efficiency of
the detection of a 22-mer RNA oligonucleotide.
In this experiment, the feasibility of a coupled, three-stage
process of the poly-(A)-polymerase reaction, reverse transcription
and PCR in the same reaction vessel should be demonstrated. For
this purpose, the coupled, three-stage process was performed with
the indicated batches under the following conditions.
As a control, the reaction was performed in a two-stage process
based on FIG. 1 B.
For the three-stage process of poly-A-reaction, reverse
transcription and PCR (PAP+RT reaction+PCR), the materials from
Table 40 were put together as indicated in Table 41.
TABLE-US-00040 TABLE 40 Materials for the Poly A Reaction, Reverse
Transcription and PCR Poly (A) Polymerase Epicenter
Biotechnologies; Material Number 400 U: PAP5104 QuantiTect
Multiplex RT-PCR Kit Qiagen; Material Number 200 rxns: 204643.
Adenosine 5'-Triphosphate (rATP) Amersham; Material Number, 25
.mu.mol: 27- 2056-01 dNTP Mix (ATGC, 10 mmol each) Amersham;
Material Number Qiagen Intern 1007430 RNase Inhibitor Promega;
Material Number: N2511 Uni GAP dT Primer 5'-TGG AAC GAG ACG ACG ACA
GAC CAA GCT TCC CGT TCT CAG CCT TTT TTT TTT TTT TTT TTT TTY VN-3'
(SEQ ID NO: 2) Com RNA From 1 g of Ground Com Husks with Qiagen
RNeasy Mini Kit (Cat. No. 74106) Plant Protocol, 10-Fold Upscale
(Maxi Shredder and Column) See Above mleu7a Oligonucleotide 5'-VGA
GGU AGU AGG UUG UAU AGU U-3' (SEQ ID NO: 2) let 7short (Specific
miRNA Primer) 5'-GAG GTA GTA GGT TGT ATA G-3' (SEQ ID NO: 1) Hum
Uni Primer 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO: 4)
GAPDH-TM-HEX_BHQ 5'HEX-CAA GCT TCC CGT TCT CAG Poly A RNA CC-BHQ 3'
(SEQ ID NO: 3) Amersham Random N8 Primer Biosciences Material
Number 27-4110-01, Oligo dT 12 Dissolved with 25 .mu.g/.mu.l in
RNase-Free Water NNNNNNNN TTTTTTTTTTTT-3' (SEQ ID NO: 20)
Phosphate
TABLE-US-00041 TABLE 41 PAP + RT Reaction + PCR with Batches in
QuantiTect Multiplex RT- PCR Master Mix (Qiagen) Final Reagents
Concentration 2x QuantiTect Multiplex RT-PCR Master Mix 1 x rATP,
10 mmol 100 .mu.mol Poly A Polymerase, 4 U/.mu.l 1 U dNTP Mix
(ATGC, 10 mmol each) 0.5 mmol Uni GAP dT Primer, 10 .mu.mol 0.05
.mu.mol RNase Inhibitor, 40 U/.mu.l 10 U QuantiTect Multiplex RT
Mix 0.2 .mu.l Hum Uni Primer, 10 .mu.mol 0.5 .mu.mol let 7short
(Specific miRNA Primer) 0.5 .mu.mol GAPDH-TM-HEX_BHQ 0.2 .mu.mol
RNase-Free Water Variable Poly A RNA, 25 .mu.g/.mu.l 10 ng/.mu.l N8
Random Primer 0.05 .mu.mol Oligo dT12 5 .mu.mol mleu7 a 10''9
Copies/.mu.l 2 .mu.l (2 .times. 10.sup.9 Copies) Corn RNA, 20
ng/.mu.l 2 .mu.l (40 ng) Total Volume 20 .mu.l
The reactions were tested three times apiece. For this purpose, the
reagents from Table 40 were pipetted together as indicated in Table
41, and the reaction was performed as indicated in Table 42.
TABLE-US-00042 TABLE 42 Reaction Protocol of the "3-in-1" Reaction
Poly A Reaction and 45 Minutes, 37.degree. C. Reverse Transcription
15 Minutes, 50.degree. C. PCR Initial Reactivation 15 Minutes,
95.degree. C. Denaturation 15 Seconds, 94.degree. C. 45x
Annealing/Extension 30 Seconds, 52.degree. C.
The reaction protocol first consisted of conditions for the
combined reaction of poly-A polymerase and reverse transcription
with the QuantiTect Multiplex Reverse Transcriptase Mix (45
minutes, 37.degree. C., and 15 minutes, 50.degree. C.). From this
followed an incubation for 15 minutes at 95.degree. C., with the
purpose of inactivating the poly-A-polymerase and reverse
transcriptase and activating the HotStarTaq DNA polymerase that is
contained in the QuantiTect Multiplex RT-PCR Master Mix. 45 PCR
cycles followed for 15 seconds at 94.degree. C. and for 30 seconds
at 52.degree. C. (see Table 43) to amplify the generated let7a-cDNA
in a real-time PCR. For detection, a fluorescence-labeled probe
specific to the 5'-tail of the Uni Gap dT primer was used. The
acquisition of the fluorescence data was carried out during the
52.degree. C. annealing/extension step. The "3-in-1" reaction was
performed with an Applied Biosystems 7500 Fast Real-Time PCR System
(Applied Biosystems) in a reaction volume of 20 .mu.l.
As shown in FIG. 14, the "3-in-1" reaction allows the specific
detection of the synthetic 22mer RNA oligonucleotide in the
background of com RNA. The sample, which contains the synthetic
22mer RNA oligonucleotide in the background of com RNA, supplies a
Ct value of 20.61, whereby the control reaction with com RNA
yielded a Ct value of 309.65. This experiment shows that the
"3-in-1" reaction can technically be used under the given
conditions.
Example 8
Implementation of the "3-in-1" process according to the invention
with use of a manual PCR primer "Hot Starts," with the purpose of
promoting the reaction of the coupled, three-stage process. As a
control, the reaction was performed in a two-stage process based on
FIG. 1 B.
The reactions were put together with the materials indicated in
Table 43 as indicated in Table 44.
TABLE-US-00043 TABLE 43 Materials for the Poly A Reaction, Reverse
Transcription and PCR Poly (A) Polymerase Epicenter
Biotechnologies; Material Number 400 U: PAP5104 QuantiTect
Multiplex RT-PCR Kit Qiagen; Material Number 200 rxns: 204643
Adenosine 5'-Triphosphate (rATP) Amersham; Material Number 25
.mu.mol: 27- 2056-01 dNTP Mix (ATGC, 10 mmol each) Amersham;
Material Number Qiagen Intern 1007430 RNase Inhibitor Promega;
Material Number: N2511 Uni GAP dT Primer 5'-TGG AAC GAG ACG ACG ACA
GAC CAA GCT TCC CGT TCT CAG CCT TTT TTT TTT TTT TTT TTT TTY VN-3'
(SEQ ID NO: 2) Com RNA From 1 g of Ground Com Husks with Qiagen
RNeasy Mini Kit (Cat. No. 74106) Plant Protocol, 10-Fold Upscale
(Maxi Shredder and Column) mleu7a Oligonucleotide 5'-VGA GGU AGU
AGG UUG UAU AGU U-3' (SEQ ID NO: 1) PCR Primer: let 7short
(Specific miRNA Primer) 5'-GAG GTA GTA GGT TGT ATA G-3' (SEQ ID NO:
4) Hum Uni Primer 5'-AAC GAG ACG ACG ACA GAC-3' (SEQ ID NO: 3)
GAPDH-TM-HEX_BHQ Amersham Biosciences Material Number 27-
Additions: 4110-01, Dissolved with 25 .mu.g/.mu.l in RNase- Poly A
RNA Free Water Random N8 Primer NNNNNNNN Oligo dT 12
TTTTTTTTTTTT-3' (SEQ ID NO: 2) Phosphate
TABLE-US-00044 TABLE 44 PAP + RT Reaction + PCR in QuantiTect
Multiplex RT-PCR Master Mix (Qiagen) Final Reagents Concentration
2x QuantiTect Multiplex RT-PCR Master Mix 1 x rATP, 10 mmol 100
.mu.mol Poly A Polymerase, 4 U/.mu.l 1 U dNTP Mix (ATGC, 5 mmol
each) 0.5 mmol Uni GAP dT Primer, 10 .mu.mol 0.05 .mu.mol RNase
Inhibitor, 40 U/.mu.l 10 U QuantiTect Multiplex RT Mix 0.2 .mu.l
RNase-Free Water Variable Poly A, 25 .mu.g/.mu.l 10 ng/.mu.l N8
Random Primer 0.05 .mu.mol Oligo dT12 5 .mu.mol mleu7 a 10''9
Copies/.mu.l 2 .mu.l (2 .times. 10.sup.9 Copies) Corn RNA, 20
ng/.mu.l 2 .mu.l (40 ng) Total Volume 20 .mu.l
With the PCR primers from Table 45, a primer mix is produced, and
the required amount for respectively one reaction is pipetted in
each case into a cover of an optical cap (covers for real-time PCR
vessels, Applied Biosystems; Material Number 4323032). Then, the
cover was incubated on a heating block at 37.degree. C. for about
20 minutes until the liquid was evaporated, and thus the primer was
dried.
After the complete drying of the PCR primer in the cover, the
reagents are pipetted together as in Table 44 and added in Optical
Tubes (real-time PCR vessels, Applied Biosystems; Material Number
4316567), sealed with the pretreated optical caps, and the PCR is
performed as indicated in Table 47.
The reactions were tested three times apiece.
TABLE-US-00045 TABLE 45 Composition of Dried Oligo-Mix in the PCR
Cover Amount of Final Concentration in Oligo/rxn PCR After the Hum
Uni Primer 10 pmol 0.5 .mu.mol let 7short (Specific miRNA 10 pmol
0.5 .mu.mol Primer GAPDH-TM-HEX_BHQ 4 pmol 0.2 .mu.mol
TABLE-US-00046 TABLE 46 Reaction Protocol of the "3-in-1" Reaction
Poly A Reaction and 45 Minutes, 37.degree. C. -- Reverse
Transcription 15 Minutes, 50.degree. C. Incubation 95.degree. C., 3
Minutes Briefly Invert 8-Strip Reaction Vessel to Dissolve the
Primer, Dried in the Cover, in the Reaction Mix PCR Initial
Reactivation 12 Minutes, 95.degree. C. Denaturation 15 Seconds,
94.degree. C. 45x Annealing/Extension 30 Seconds, 52.degree. C.
The reaction protocol first consisted of conditions for the
combined reaction of poly-A polymerase and the reverse
transcription with the QuantiTect Multiplex Reverse Transcriptase
Mix (45 minutes, 37.degree. C., and 15 minutes, 50.degree. C.).
Then, the reactions were heated for 3 minutes to 95.degree. C. with
the purpose of inactivating the poly-A-polymerase and reverse
transcriptase enzymes. Then, the PCR tubes were briefly removed
from the device and inverted, with the purpose of redissolving the
dried primer that is present in the covers and making it available
for the following PCR reaction. An incubation followed from this
for 12 minutes at 95.degree. C. to activate the HotStarTaq DNA
polymerase contained in the QuantiTect Multiplex RT-PCR Master Mix.
A reactivation that is shorter by 3 minutes was selected here,
since the reaction mix was already heated previously for
inactivating the poly-A-polymerase and reverse transcriptase
enzymes for 3 minutes to 95.degree. C. 45 PCR cycles followed for
15 seconds at 94.degree. C. and 30 seconds at 52.degree. C. (see
Table 47) to amplify the generated let7a-cDNA in a real-time PCR.
For detection, a fluorescence-labeled probe specific to the 5'-tail
of the Uni Gap dT Primer was used. The acquisition of the
fluorescence data was carried out during the 52.degree. C.
annealing/extension step. The "3-in-1" reaction was performed with
an Applied Biosystems 7500 Real-Time PCR System (Applied
Biosystems) in a reaction volume of 20 .mu.l.
FIGURES
FIG. 1A-FIG. 1B shows a comparison between the one-stage process
according to this invention (B) and the two-stage process as it is
known in the prior art (A). Poly A polymerase: enzyme with
polyadenylenation activity in terms of the invention; reverse
transcriptase: enzyme with reverse transcriptase activity in terms
of the invention; rATP: ribonucleotide, here
adenosine-5'-triphosphate by way of example; dNTPs:
deoxyribonucleotides; oligo dT tail primer: anchor oligonucleotide
with various possible embodiments in terms of the invention; Uni
GAP dT primer: special embodiment of the anchor oligonucleotide;
tail: 5' tail as an optional part of the anchor oligonucleotide; w:
defines the length according to the invention of the homopolymer
tail that is attached by the polyadenylation activity (greater than
10-20 bases); x, y: defines the type and length according to the
invention of the 3'-anchor sequence of the anchor oligonucleotide
according to the invention; z: defines the length of the
homopolymer portion of the anchor oligonucleotide according to the
invention. FIG. 1 discloses "AAAAA[A].sub.1-w," as SEQ ID NO:
21.
FIG. 2 shows a graphic depiction of the Ct values from Table 6:
Condition a) contained templates in each case, and in condition b),
only H.sub.20, instead of templates, was added (H.sub.20 in PAP
reaction). In b), no signal up to PCR cycle 40 (maximum number of
the cycles performed) was obtained, therefore the indication is "No
Ct."
FIG. 3 shows an agarose gel analysis of the real-time PCR products
from Example 1. M: 100 bp ladder (Invitrogen, Catalog No.
15628-050). 2% Agarose, colored with ethidium bromide in TAE as a
running buffer. Loading diagram: Trace 1: markers, then in each
case the 3.times. determinations have been plotted next to one
another: 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b.
FIG. 4 shows a tabular depiction of Ct values that were obtained by
real-time PCR analysis of the reaction products of the batches
described in Table 8. At 3 and 6, no signal up to PCR cycle 40
(maximum number of the cycles performed) was obtained; therefore
the indication is "no Ct."
FIG. 5 shows a graphic depiction of Ct values that were obtained by
real-time PCR analysis of the reaction products of the batches
described in Table 8.
FIG. 6 shows a tabular depiction of Ct mean values that were
obtained by real-time PCR analysis of the reaction products of the
batches b) and e) described in Table 18.
In 2, 4 and 5 with standard RT, a signal was detected at the
earliest only after cycle 38 or in 2), no signal, therefore the
indication is "no Ct."
FIG. 7 shows a tabular depiction of real-time PCR results of
batches b) and d) from Table 18, as well as the controls with only
one primer from Table 19, batches a)-d). No signal up to PCR cycle
40 (maximum number of the cycles performed) was obtained; therefore
the indication is "no Ct."
FIG. 8 shows an agarose gel analysis of the real-time PCR products
from Example 3. M: 100 bp ladder (Invitrogen, catalog No.
15628-050). 2% Agarose, colored with ethidium bromide in TAE as a
running buffer.
FIG. 9 shows a tabular depiction of Ct mean values that were
obtained by real-time PCR analysis of the reaction products of the
batches 1a), b) and 2a), b) described in Table 30.
Lower Part:
Graphic depiction of Ct mean values that were obtained by real-time
PCR analysis of the reaction products of the batches described in
Table 30.
FIG. 10 shows a tabular depiction of the Ct mean values that were
obtained by real-time PCR analysis of the reaction products of the
batches described in Table 36, 1-5, and in Table 37, 6-9.
Lower Part:
Graphic depiction of Ct mean values that were obtained by real-time
PCR analysis of the reaction products of the batches described in
Table 36, 1-5 and in Table 37, 6-9.
FIG. 11 shows a list of the nucleic acid sequences used.
FIG. 12 shows anchor oligonucleotides according to the invention.
FIG. 12 discloses "poly(T)15-50" as SEQ ID NO: 22 and "Anchor
Oligonucleotides" as SEQ ID Nos: 23-26, 26-27 and 10-19,
respectively, in order of appearance.
FIG. 13A-FIG. 13B shows a comparison between the one-stage "3-in-1"
process according to this invention (B) and the three-stage process
as it is known in the prior art (A). Poly A polymerase: enzyme with
polyadenylation activity in terms of the invention; (FIG. 13
discloses "AAAA[A].sub.1-w," as SEQ ID NO: 21).
Reverse transcriptase: enzyme with reverse transcriptase activity
in terms of the invention;
rATP: ribonucleotide, here adenosine-5'-triphosphate by way of
example;
dNTPs: deoxyribonucleotides;
Oligo dT tail primer: anchor oligonucleotide with various possible
embodiments in terms of the invention;
Uni GAP dT primer: special embodiment of the anchor
oligonucleotide; Tail: 5'-Tail as an optional part of the anchor
oligonucleotide;
w: defines the length, according to the invention, of the
homopolymer tails that are attached by the polyadenylation activity
(greater than 10-20 bases);
x, y: defines the type and length, according to the invention, of
the 3'-anchor sequence of the anchor oligonucleotide according to
the invention;
z: defines the length of the homopolymer portion of the anchor
oligonucleotide according to the invention.
PCR primer: at least one oligonucleotide for specific detection of
the cDNA species, optionally at least one probe;
PCR enzyme: enzymatic activity that allows the specific detection
of the cDNA species contained in the sample.
FIG. 14 shows a tabular depiction of the Ct mean values of a
"3-in-1" reaction, i.e., the combined poly-(A)-polymerase reaction,
reverse transcription and real-time PCR analysis coupled in a
reaction vessel, according to the reaction batch from Example 7
corresponding to Table 41 and the reaction batch from Table 42.
Lower Part:
Graphic depiction of Ct mean values of a "3-in-1" reaction, i.e.,
the combined poly-(A)-polymerase reaction, reverse transcription
and real-time PCR analysis coupled in a reaction vessel, according
to the reaction batch from Example 7 corresponding to Table 41 and
the reaction batch from Table 42.
FIG. 15 shows a tabular depiction of Ct mean values of a "3-in-1"
reaction, i.e., the combined poly-(A)-polymerase reaction, reverse
transcription and real-time PCR analysis coupled in a reaction
vessel, after the reaction batch of Example 8 corresponding to
Table 44 and the reaction batch from Table 46.
Lower Part:
Graphic depiction of Ct mean values of a "3-in-1" reaction, i.e.,
the combined poly-(A)-polymeras reaction, reverse transcription,
and real-time PCR analysis coupled in a reaction vessel.
FIG. 16 shows various amounts (10 pg to 1 .mu.g) of miRNAeasy RNA,
which were reverse transcribed with use of miScript in the presence
or absence of 100 ng of poly-(A) or poly-(C). The thus produced
cDNA was used in a real-time PCR; in this case, miR-16 and let-7a
were tested.
FIG. 17 shows various amounts (10 pg to 1 .mu.g) of miRNAeasy RNA,
which were reverse transcribed with use of miScript in the presence
or absence of various amounts of poly(A). The thus produced cDNA
was used in a real-time PCR; in this case, miR-16 was tested.
FIG. 18 shows various amounts (10 pg to 1 .mu.g) of miRNAeasy RNA,
which were reverse transcribed with use of miScript in the presence
or absence of various amounts of poly-(C). The thus produced cDNA
was used in a real-time PCR; in this case, miR-16 was tested.
FIG. 19 shows the use of 10 pg of miRNeasy RNA, which was reverse
transcribed in the presence or absence of 50 ng of poly-(C) with
use of the miScript RT Kit. The thus produced cDNA was used in a
real-time PCR to detect GAPDH.
FIG. 20 shows various amounts (1-100 ng) of miRNeasy RNA, which
were reverse transcribed with use of miScript in the presence or
absence of various amounts of poly-(C). The thus produced cDNA was
used in a real-time PCR, in this case to test GADPH.
FIG. 21 shows the use of 10 and 100 pg of miRNeasy RNA, which were
reverse transcribed in the presence or absence of 50 ng of poly-(C)
and with use of the miScript RT Kit. The thus produced cDNA was
tested in a real-time PCR to detect GAPDH.
FIG. 22 shows various amounts (1-100 ng) of miRNeasy RNA, which
were reverse transcribed with use of miScript in the presence or
absence of various amounts of poly-(C). The thus produced cDNA was
used in a real-time PCR, in this case to test CDC2.
FIG. 23 shows the use of 1 ng of miRNeasy RNA, which was reverse
transcribed in the presence or absence of 50 ng of poly-(C) and
with use of the miScript RT Kit. The thus produced cDNA was tested
in a real-time PCR to detect CDC2.
SEQUENCE LISTINGS
1
27122RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1ugagguagua gguuguauag uu
22265DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2tggaacgaga cgacgacaga ccaagcttcc
cgttctcagc cttttttttt tttttttttt 60ttvvn 65318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3aacgagacga cgacagac 18419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4gaggtagtag gttgtatag 19518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5tggctcagtt cagcagga 18619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6tagcagcaca taatggttt 19719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7tagcagcacg taaatattg 19829DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8gtacactgac ttgagaccag ttgaataaa 29920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9caagcttccc gttctcagcc 201074DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10tggaacgaga cgacgacaga ccaagcttcc cgttctcagc
cttttttttt tttttttttt 60tttttttttt tvvn 741150DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11aacgagacga cgacagactt tttttttttt tttttttttt
ttttttttvn 501249DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 12aacgagacga cgacagactt
tttttttttt tttttttttt ttttttttv 491349DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13aacgagacga cgacagactt tttttttttt tttttttttt
ttttttttn 491450DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 14aacgagacga cgacagactt
tttttttttt tttttttttt ttttttttnn 501551DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15aacgagacga cgacagactt tttttttttt tttttttttt
ttttttttvn n 511652DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 16aacgagacga cgacagactt
tttttttttt tttttttttt ttttttttvn nn 521751DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17aacgagacga cgacagactt tttttttttt tttttttttt
ttttttttnn n 511873DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 18tggaacgaga cgacgacaga
ccaagcttcc cgttctcagc cttttttttt tttttttttt 60tttttttttt tvn
731974DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 19tggaacgaga cgacgacaga ccaagcttcc
cgttctcagc cttttttttt tttttttttt 60tttttttttt tvnn
742012DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 20tttttttttt tt 122125DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21aaaaaaaaaa aaaaaaaaaa aaaaa 252250DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 502367DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 23nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnntttttt tttttttttt tttttttttt 60ttttvvn
672466DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 24nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnntttttt tttttttttt tttttttttt 60ttttvn 662565DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 25nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnntttttt
tttttttttt tttttttttt 60ttttv 652633DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26tttttttttt tttttttttt tttttttttt vvn
332767DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 27nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnntttttt tttttttttt tttttttttt 60ttttvnn 67
* * * * *